Segmented micro rna mimetics

ABSTRACT

This invention relates generally to segmented oligonucleotides capable of modulating gene expression. Specifically, the instant invention relates to segmented microRNA (miRNA) oligonucleotides, including segmented miRNA precursors and segmented pre-microRNAs. The invention also relates to compositions comprising such segmented oligonucleotides, as well as to methods of making and using such oligonucleotides for diagnosis and treatment of diseases associated or causally linked to aberrant levels or activities of gene expression, including aberrant levels of coding and/or non-coding RNA.

PRIORITY APPLICATIONS

This application claims the benefit of U.S. Patent Application No. 61/236,486 filed Aug. 24, 2009.

TECHNICAL FIELD

This invention relates generally to segmented oligonucleotides capable of modulating gene expression. Specifically, the instant invention relates to segmented microRNA (miRNA) mimetic oligonucleotides, including segmented miRNA precursors and segmented pre-microRNAs. The invention also relates to compositions comprising such segmented oligonucleotides, as well as to methods of making and using such oligonucleotides for diagnosis and treatment of diseases associated or causally linked to aberrant levels or activities of gene expression, including aberrant levels of coding and/or non-coding RNA.

BACKGROUND

Segmented oligonucleotides based on short interfering RNA (siRNA) have been evaluated for RNA interference (RNAi) activity. Leuschner et al., (2006 EMBO 7:314) described an RNA-induced silencing complex which has a discontinued passenger (or sense) strand and a 2′-O-methyl modified nucleotide at position 9 of the passenger strand (5′ to 3′), the natural cleavage site. Bramsen et al., (2007 Nucleic Acids Res. 35:5886) described an RNAi-active siRNA molecule comprising an internally segmented passenger strand, where the nick or gap is not necessarily located at the natural cleavage site, stabilized with locked nucleic acid (LNA) modifications at a number of positions. See also, e.g., Wengel et al., PCT Publication WO 2007/107162 A2; Quay et al., PCT Publication WO 2008/049078. None of Leuschner, Bramsen, Wengel and Quay described RNAi-active molecules having discontinued guide (or antisense) strands. In fact, Bramsen and Wengel indicated that duplexes designed to contain discontinuities in the guide strands completely eliminated silencing of the target.

The mechanistic difference between miRNA-mediated RNAi and siRNA-mediated RNAi can make certain modifications and/or designs suitable for one but not the other. Thus, there remains a heightened interest in formulating new and advantageous design features suitable for miRNA mimetics.

SUMMARY OF THE INVENTION

The specification describes certain segmented double-stranded miRNA mimetics having at least one non-contiguous (or discontinuous) strand comprising a miRNA sequence, which can be introduced or applied to cells, tissues, and/or organisms to mediate RNAi. These molecules are referred to herein as segmented miRNA mimetics, which comprise a guide strand and a passenger strand. The guide strand comprises at least two contiguous stretches of nucleotides separated by a discontinuity. The passenger strand can be fully contiguous, or alternatively can also comprise at least two contiguous stretches of nucleotides separated by a discontinuity. Segmented miRNA mimetics of the invention therefore have at least one non-contiguous guide strand comprising one or more miRNA sequences, or a portion thereof, including the seed sequence of such miRNA sequences. Non-limiting examples of miRNA sequences are those selected from the miRBase as of the filing date of the present invention; see for example Griffiths-Jones (2006) miRBase: the microRNA sequence database. Methods in Molecular Biology 342: 129-138 and miRBase release 13.0; http://micrirna.sanger.ac.uk/. A segmented miRNA of the invention therefore can include one or more miRNA sequences selected from SEQ ID NOs: 1-1090 of Table I, including portions thereof, such as the seed sequences.

A segmented miRNA mimetic of the invention comprises at least one discontinuity in the guide strand, and optionally at least one discontinuity in the passenger strand that can be the same or different as the discontinuity in the guide strand. Such discontinuities include nicks, gaps, substitutions, and/or insertions. Segmented miRNA mimetic can comprise mixtures of different discontinuities in one or both strands.

A segmented miRNA mimetic of the invention comprises about 12 to about 26 nucleotides in each strand, and further comprises about 10 to about 26 base pairs between the strands. Thus, a prototypical segmented miRNA mimetic of the invention generally comprises two strands having complementarity to form a duplex, each strand having between about 12 to about 26 nucleotides, wherein the guide strand comprises any of SEQ ID NOs: 1-1090 or a portion thereof, and wherein the guide strand further comprises at least on discontinuity.

Segmented miRNA mimetics of the invention can be administered to a cell, a tissue or an organism to supplement or increase the levels of their corresponding endogenous miRNAs and hence potentiate RNAi activity against their corresponding miRNAs targets. Because each endogenous miRNA typically has multiple targets, an exogenously introduced segmented miRNA mimetic of the invention does not necessarily share the same number, identity or type of targets with its corresponding endogenous miRNA. However, the exogenously-introduced segmented miRNA mimetic exerts activity on at least one (i.e., one or more or all) of the targets of its corresponding endogenous miRNA.

A segmented miRNA mimetic can be chemically modified at the nucleic acid base, phosphodiester backbone, or sugar to achieve, for instance, increased stability and/or reduced immunogenicity, and other pharmaceutically desirable attributes, including properties that would allow for enhanced delivery or lower toxicity. Methods of chemically modifying oligonucleotides to achieve such ends are known in the art. For instance, numerous such methods are set forth in McSwiggen, et al., U.S. Publication No. 2006/0211642.

In a further aspect, the specification provides a composition comprising one or more (i.e., in the number of individual molecules and/or in types) segmented miRNA mimetics in a pharmaceutically acceptable carrier or diluent. In another aspect, the specification provides a method of introducing or applying one or more segmented miRNA mimetics to cells (regardless of whether the RNAi or other gene modulation process takes place inside the cells, outside the cells, or on the cell-membrane), tissues, organisms, or reconstituted in vitro systems, to increase the levels of corresponding endogenous miRNAs. Embodiments of the invention include methods of modulating gene expression, biologic pathways, or physiologic pathways in cells, cultures, tissues, or organisms such as subjects or patients, comprising administering one or more segmented miRNA mimetics of the invention in an amount that is sufficient to modulate the expression of one or more genes that are regulated by the corresponding endogenous miRNAs. In a specific embodiment, more than one type of segmented miRNA mimetic is administered. For example, a number of different segmented miRNA mimetics of the invention can be administered concurrently, in sequence, or in an ordered progression.

In certain embodiments, administration of the composition(s) can be enteral or parenteral. In certain aspects, enteral administration is oral. In further aspects, parenteral administration is intralesional, intravascular, intracranial, intrapleural, intratumoral, intraperitoneal, intramuscular, intralymphatic, intraglandular, subcutaneous, topical, intracronchial, intratracheal, intranasal, inhaled, or instilled. Compositions of the invention can be administered regionally or locally, and not necessarily directed into a lesion.

Embodiments of the invention can include obtaining or assessing a gene expression profile or miRNA profile of a target cell, tissue, or organism prior to selecting the mode of treatment, by, for example, administration of one or more segmented miRNA mimetics. In certain aspects of the invention, one or more segmented miRNA mimetics can modulate a single gene. In a further aspect, one or more genes in one or more genetic, cellular, or biologic/physiologic pathways can be modulated by a single segmented miRNA mimetic or a complement thereof, alone or in combination with other miRNAs or mimetics, or with other nucleic acid-based gene modulators, such as siRNAs, antisense molecules, ribozyme molecules, and the like.

A further aspect of the invention is directed to a method of modulating a cellular pathway comprising administering to the cell an amount of a segmented miRNA mimetic, alone or in combination with other miRNAs, mimetics, siRNAs, or other suitable nucleic-acid based or non-nucleic acid based agents capable of modulating one or more relevant genes in the same or associated pathways. In a related aspect, the invention is directed to methods of modulating a cellular pathway comprising administering to the cell a segmented miRNA mimetic in an amount sufficient to modulate the gene expression, function, status, or state of a cellular pathway, in particular a pathway that is known to include one or more genes associated with the corresponding endogenous miRNA. Modulation of a cellular pathway includes, but is not limited to, modulating the expression of one or more genes associated with the pathway. Modulation of a gene includes inhibiting its function, also called “down-regulate a gene,” or providing an agonist to augment its functional, also called “up-regulating a gene.” What is modulated is either the expression level or activity of a gene or its related gene product or protein.

Compositions and methods comprising a segmented miRNA mimetic are also useful for treating diseases or disorders associated with aberrant expression levels or activity of one or more corresponding miRNA targets. These diseases and/or disorders include, for example, hyperproliferative disorders (e.g., cancer), inflammatory conditions (e.g., arthritis), respiratory diseases, pulmonary diseases, cardiovascular diseases, autoimmune diseases, allergic disorders, neurologic diseases, infectious diseases (e.g., viral infections), renal diseases, transplant rejections, or any other conditions that respond to such modulation.

Still a further embodiment includes methods of treating a patient with a pathological condition comprising one or more steps: (a) administering to the patient an isolated or a synthetic segmented miRNA mimetic of the invention in an amount sufficient to modulate the expression of a cellular pathway; and (b) administering a second therapy, wherein the modulation of the cellular pathway in (a) sensitizes the patient to the second therapy. A cellular pathway can include, but is not limited to, one or more pathways that are known to be associated with known miRNAs listed in the miRBase as of the date of filing of the instant application. A second therapy can include administration of one or more miRNAs or mimetics targeting the same or different mRNAs, or one or more other therapeutic nucleic acids. A second therapy can also be one selected from other standard therapies, such as chemotherapy, radiation therapy, drug therapy, immunotherapy, and the like.

The invention also features a kit or article of manufacture comprising one or more segmented miRNA mimetics, typically in a pharmaceutical composition, and instructions for administering the composition to treat a pathological condition. Optionally, the kit or article of manufacture can contain one or more other pharmaceutical compositions or agents and instructions for their use in conjunction with the pharmaceutical composition comprising the segmented miRNA mimetics.

In yet a further aspect of the invention, one or more segmented miRNA mimetics of the invention can be included in a kit or article of manufacture for assessment or diagnosing of a pathological condition or the risk of developing a pathological condition.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments can be combined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 illustrate examples of different structural features of segmented miRNA mimetics. It will be apparent to the skilled person in the art that the various structural features illustrated in these figures can be combined. For example, a particular pattern of the overhangs can be combined with a molecule comprising one or more discontinuities at particular positions, which can or can not be linked, by phosphodiester bonds or non-phosphodiester connectors. Therefore, these figures should be interpreted as merely representative but non-limiting, such that, for example, the nature and position of the discontinuities can be changed and additional features, such as, for example, overhangs and nucleotide analogs, can be introduced to the same molecules.

FIG. 1 illustrates the basic structural features of a segmented miRNA mimetic. A shows 3 representative segmented miRNA mimetics, comprising nicks or gaps in the guide (the lower strand), or in both the passenger (upper strand) and the guide strands. Each box represents a nucleotide (including a nucleotide analog), or a non-nucleotide substitute moiety. Each line connecting the boxes represents a phosphodiester bond or non-phosphodiester connector. Distinct contiguous stretches of nucleotides in a given strand are colored/shaded differently to facilitate the identification of the nick or gap positions. B shows a representative conformation of a segmented miRNA mimetic, wherein the passenger strand is uninterrupted and wherein the guide strand comprises a gap of 1 nucleotide. A similar motif comprising a nick instead of a gap is contemplated by the instant invention. C shows a representative segmented miRNA mimetic wherein each of the passenger strand and the guide strand comprises a nick. Some of the discontinuities in this figure are shown as nicks. However, those skilled in the art would appreciate that gaps can be present at similar positions, and other types of discontinuities such as substitutions and insertions are also contemplated. Moreover, those skilled in the art would appreciate that a gap can be a stretch of 1 to about 10 contiguous vacant nucleotide positions.

FIG. 2A illustrates various overhangs and blunt ends can be utilized in the design of a segmented miRNA mimetic of the invention. The discontinuities are shown in this figure as nicks or gaps of a single nucleotide, but other kinds of discontinuities, including larger gaps of one or more (e.g., up to 10) nucleotides, as well as substitutions and/or insertions are also contemplated. FIG. 2B illustrates a representative segmented miRNA mimetic wherein one set of the terminal ends are connected by a linker.

FIG. 3 illustrates that one or more discontinuities (nicks or gaps are shown, but also substitutions and insertions) can be present in the guide strand, or in both the passenger and the guide strands.

FIG. 4 illustrates that the sizes or positions of the discontinuities on the guide strand, or both the passenger and guide strands can vary. The molecules are named according to the starting and ending positions of the discontinuities from the 5′-end.

FIG. 5 illustrates that one or more suitable non-nucleotide substitutions or insertions can be used to connect a pair of neighboring contiguous stretches of nucleotides in the passenger strand, the guide strand, or both the passenger and guide strands.

FIG. 6 illustrates certain segmented miRNA mimetics comprising inverted abasic modifications at the internal ends. The molecules are named according to the starting and ending positions of the discontinuities.

FIGS. 7-20 show experimental data obtained from the examples herein.

FIG. 7 illustrates RNAi activity against an endogenous miR-124 target, CD164. RNAi activity of various segmented miRNA mimetics derived from miR-124 (“segmented miR-124”) was measured. Levels of knockdown achieved by the segmented miR-124 constructs and by the corresponding non-segmented miR-124 mimetic, comprising the endogenous mature miR-124 sequence as its guide strand, were determined. The segmented miR-124 constructs can comprise one or more locked nucleic acid (“LNA”) nucleotides in one or both strands, each represented by an underlined nucleotide in Table III herein, although the LNAs are not necessarily placed at the underlined nucleotide positions.

FIG. 8 illustrates RNAi activity against another endogenous miR-124 target, VAMP3. RNAi activity of various segmented miR-124 mimetics was measured. Levels of knockdown achieved by the segmented miR-124 constructs and by the corresponding non-segmented miR-124 mimetic, comprising the endogenous mature miR-124 sequence as its guide strand, were determined. The segmented miR-124 constructs can comprise one or more LNA nucleotides in one or both strands, each represented by an underlined nucleotide in Table IV herein, although the LNAs are not necessarily placed at the underlined nucleotide positions.

FIG. 9A illustrates RNAi activity of various segmented miR-124 mimetics (i.e., of Table V) against CD164. Levels of knockdown achieved by the segmented miR-124 constructs and by the corresponding non-segmented miR-124 mimetic, comprising the endogenous mature miR-124 sequence as its guide strand, were determined. Knockdown or inhibition, or the lack thereof, by segmented miR-34 constructs, which were designed based on human miR-34, is presented as a negative control.

FIG. 9B illustrates RNAi activity against an endogenous miR-34 target, TK1, of various segmented miRNA mimetics derived from miR-34 (“segmented miR-34”) (i.e., molecules of Table V). Levels of knockdown achieved by the segmented miR-34 constructs and by the corresponding non-segmented miR-34 mimetic, comprising the endogenous mature miR-34 sequence as its guide strand, were determined. Knockdown or inhibition, or the lack thereof, by segmented miR-124 constructs, is presented as a negative control.

FIG. 10A illustrates RNAi-mediated activity against CD164 by segmented miR-124 constructs comprising one or more inverted abasic modified internal ends (i.e., molecules of Table VI). FIG. 10B illustrates RNAi-mediated activity against VAMP3 by segmented miR-124 constructs comprising one or more inverted abasic modified internal ends.

FIG. 11A illustrates RNAi-mediated activity against CD164 by segmented miRNA-124 constructs comprising abasic substitutions (i.e., molecules of Table VII). FIG. 11B illustrates RNAi-mediated activity against VAMP3 by segmented miR-124 constructs comprising abasic substitutions.

FIG. 12 demonstrates that miR-124 activity tolerates segmentation of the passenger or guide strands. FIG. 12A includes schematic illustrations of miR-124 duplex designs, wherein the top strand represents the passenger strand and the bottom strand represents the guide strand. The gray-shade change indicates the site of a break in the strand backbone. The dark gray circle in the G10Cy3.12/P schematic represents a 5′-Cy3 label, and the light gray circle in the G10i.12/P schematic indicates a 5′ inverted abasic nucleotide. FIG. 12B illustrates the dose-dependent response of CD164, a known miR-124 target, to various concentrations of the designs shown in FIG. 12A in HCT-116 cells as measured by RT-qPCR. UC3 corresponds to the negative control oligomer. EC50s for each curve are as follows: G/P, 0.12 nM; G/P10.12, 0.29 nm; G10.12/P, 0.22 nM; G10.11/P, 0.53 nM; G10i.12/P, 0.21 nM; G10Cy3.12/P, 0.45 nm.

FIG. 13 provides a microarray analysis showing seed-based activity from a segmented guide strand. FIG. 13A is a graphic illustration of the microarray signature 24 hours after transfection of 10 nM G10.12/P, plotted as expression ratio (relative to mock transfection) versus fluorescence intensity. Significantly down regulated probes (P<1e-6) are seen as gray data points below the black data points and unregulated probes are seen as gray data points above the black data points. Hypergeometric analysis of the hexamer content of the down regulated UTRs showed that the most significantly enriched hexamer (P<1e-20) was GCCTTA, corresponding to positions 2-7 of the transfected miR-124. FIG. 13B provides a comparison of gene expression data from G10.12/P transfected cells and G/P transfected cells. Expression ratio of the G/P transfection versus the mock transfection is plotted on the x-axis and the G10.12/P expression ratio versus mock transfection on the y-axis. The weighted correlation coefficient was calculated as 0.9, illustrating the similar effects of both RNA complexes on gene expression.

FIG. 14 illustrates that the activity of an unsegmented miR-124 guide strand is enhanced by additional complementarity beyond the seed sequence, but activity of a segmented guide strand is not. FIG. 14A is a schematic of miR-124 target sites that were duplicated and inserted into dual luciferase reporter vectors. FIG. 14B illustrates that G/P miR-124 exhibits suppression of activity from a reporter with a seed sequence match, which is enhanced by reporters containing additional complementarity to the 3′ end of the guide strand (2×7a3p) or full-length complementarity. The EC50s for the curves: 2×7a, 0.39 nM; 2×7a3p, 0.08 nM; 2×FL, 0.09 nM. The EC50s for the curves are: 2×7a, 0.41 nM; 2×7a3p, 0.06 nM; 2×FL, 0.09 nM. FIG. 14C illustrates that segmentation of the passenger strand preserves the trends seen in FIG. 14B. FIG. 14D shows that activity from reporters with 3′ complementarity is identical to that observed from the seed-only reporter when the guide strand is segmented. The EC50s for the curves are: 2×7a, 0.89 nM; 2×7a3p, 0.61 nM; 2×FL, 0.80 nM.

FIG. 15 illustrates RNAi activity mediated by a segmented miR-124 precursor. A 58-mer miR-124 precursor was designed and transfected into HCT-116 cells at varying concentrations. FIG. 15A is a miR-124 precursor schematic. FIG. 15B is a graph showing CD164 knockdown as measured by RT-qPCR. Activity of the unsegmented precursor is comparable to that of the mature miRNA, while activity of the segmented precursor is reduced but is significantly above background. The EC50s for the curves are: G/P, 0.14 nM; hairpin precursor, 0.31 nM; segmented precursor, 1.48 nM.

FIG. 16 illustrates that a segmented guide strand exhibits decreased knockdown for targets containing 3′ supplementary pairing. Microarray data shown in FIG. 13B was further analyzed, specifically to analyze 1057 downregulated genes that contain TargetScan seed matches. The TargetScan 3′ pairing score was then calculated for these genes, and the downregulation of the top quartile of 3′ scores (containing a high degree of 3′ pairing) was compared with the downregulation of the bottom quartile of 3′ scores. The cumulative distribution of the difference in downregulation (between G/P and G10.10/P) is plotted for the top and bottom quartiles. A Kolmogorov-Smirnov test (p=0.02) shows that the top quartile of genes shows less knockdown in G10.10/P versus G/P, as compared with the bottom quartile.

FIG. 17A illustrates knockdown of CD164 expression by segmented miR-124 mimetics comprising deletions and c3 or c6 substitutions, while FIG. 17B illustrates knockdown of VAMP3 expression by these segmented microRNAs.

FIG. 18A illustrates knockdown of CD164 expression by segmented miR-124 comprising c3 substitutions, while FIG. 18B illustrates knockdown of VAMP3 expression by these segmented microRNAs.

FIG. 19A illustrates knockdown of CD164 expression by segmented miR-124 comprising c6 substitutions, while FIG. 19B illustrates knockdown of VAMP3 expression by these segmented microRNAs.

FIG. 20A illustrates knockdown of CD164 expression by segmented miR-124 comprising c3 and c6 insertions, while FIG. 20B illustrates knockdown of VAMP3 expression by these segmented microRNAs.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

“About” as used herein indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.

“Analog” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a compound that is structurally similar to a parent compound (e.g., a nucleotide), but differs in composition (e.g., one or more atom(s) or functional group(s) is/are different, added, or removed). The analog can have different chemical or physical properties than the original parent compound and can have improved biological or chemical activity. For example, the analog can be more hydrophilic or it can have altered activity of the parent compound. The analog can be a naturally or non-naturally occurring (e.g., chemically-modified or recombinant) variant of the original parent compound. An example of an RNA analog is an RNA molecule comprising a nucleotide analog. An example of a nucleotide analog is a nucleotide that is chemically modified at the sugar, base or nucleoside, as is generally known in the art.

The term “aptamer” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule wherein the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, e.g., Gold et al, 1995 Annu. Rev. Biochem. 64:163; Brody and Gold, 2000 J. Biotechnol. 74:5; Sun, 2000 Curr. Opin. MoI. Ther. 2: 100; Kusser, J. 2000 Biotechnol. 74:21; Hermann and Patel, 2000 Science 257:820; and Jayasena, 1999 Clinical Chem. 45:1628).

As described herein, a “base pair” can be formed between two nucleotides, a nucleotide and a modified nucleotide, two modified nucleotides, a nucleotide and a nucleotide analog, two nucleotide analogs, a nucleotide and a non-nucleotide substitute moiety, or two non-nucleotide substitute moieties. In a specific embodiment, a non-nucleotide substitute can comprise any chemical moiety that is capable of associating with a component of the cellular RNAi machinery, such as, for example, the PAZ domain, the PIWI domain, and/or other Argonaute protein domains associated with the RISC. Non-traditional Watson-Crick base pairs are also understood as “non-canonical base pairs,” which is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+ carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

The term “biodegradable” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.

The term “biodegradable linker” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, connecting a biologically active molecule to a segmented miRNA mimetic of the invention or to either the passenger and/or guide strands of a segmented miRNA mimetic of the invention. The biodegradable linker can be attached to a segmented miRNA mimetic of the invention at one or more of the terminal ends, internal ends, or any other nucleotide positions that is not vacant. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-β-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

The term “biologically active molecule” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, guide nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siRNA, miRNA mimetics, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.

By “capable of participating in RNAi against endogenous RNA targets of their corresponding naturally-occurring miRNAs” is meant that, when RNAi activity is measured by a suitable in vivo or in vitro assay or method, a segmented miRNA mimetic molecule of the invention demonstrates at least 5% or more of the knockdown effect against a target of its corresponding naturally-occurring miRNA as compared to the knockdown effect achieved by a non-segmented miRNA mimetic molecule directed to the same target under same experimental conditions. Preferably, a segmented miRNA mimetic molecule of the invention is capable of achieving 25% or more, 35% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or even 100% or more (i.e., equal or more potent RNAi activity) knockdown of the target than a non-segmented miRNA mimetic against the same target.

The term “cap structure” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to chemical modifications, which have been incorporated into the ends of oligonucleotide (see, for example, Matulic-Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications can protect certain nucleic acid molecules from exonuclease degradation, and can impart certain advantages in delivery and/or cellular localization.

As used herein, the term “complementary” (or “complementarity”) refers to its meaning as is generally accepted in the art. The term generally refers to nucleic acid sequences that are capable of base-pairing according to the standard Watson-Crick complementarity rules, that is purines will base pair with pyrimidines to form combinations: guanine paired with cytosine (G:C); and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Base-pairing according to the Standard Waston-Crick complementarity rules can include base pairs formed between modified or nucleotide analogs. Aside from forming hydrogen bond(s) with each other according to the traditional Waston-Crick rules, a nucleic acid sequence can form other non-traditional types of base pairing with another nucleic acid sequence, and as such, the two nucleic acid sequences can also be called “complementary.” As used herein, the term “complementary” thus encompasses any “base-pairing,” which can be by hydrogen bonds or by any other interactions, of nucleotides, modified nucleotides, analogs, and/or non-nucleotides that provide sufficient binding free energy between the strands to allow the relevant function of the segmented miRNA mimetic, e.g., RNAi activity, to proceed. Determination of binding free energies for nucleic acid molecules is known in the art (see, e.g., Turner et al., 1987 CSH Symp. Quant. Biol. LII:123; Frier et al., 1986 Proc. Nat. Acad. Sci. USA 83:9373; Turner et al., 1987 J. Am. Chem. Soc. 109:3783).

A percent complementarity indicates the percentage of contiguous residues in a first nucleic acid molecule that can form hydrogen bonds (e.g., in Watson-Crick base-pairing) with a second nucleic acid sequence. For example, a first nucleic acid molecule can have 10 nucleotides and a second nucleic acid molecule can have 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules, which can or can not form a contiguous double-stranded region, represents 50%, 60%, 70%, 80%, 90%, or 100% complementarity, respectively. Complementarity can be found between two regions of a same nucleic acid molecule, such as, for example, in a hairpin loop or a stem loop structure. In other embodiments, complementarity can be found between two different nucleic acid molecules, such as, for example, in a segmented miRNA mimetic of the invention comprising distinct and separate passenger and guide strands.

In keeping with the usual practice by those of ordinary skill in the art, when the passenger strand and guide strand of the corresponding non-segmented miRNA are aligned on paper, (with the passenger strand arranged from 5′ to 3′ (left to right) and the guide strand arranged from 3′ to 5′ (left to right)) such that the each pair of complementary (base-pairing) nucleobases are located at directly opposite positions in the passenger and guide strand, the relative positions of the base-pairing nucleotides are termed “complementary nucleotide positions.” It is often helpful to mark the position of the nucleotides in the non-segmented miRNA mimetic and use those positions to mark nicks, gaps, substitutions, or insertions introduced into a corresponding segmented mimetic construct. Typically the first nucleotide position at the 5′-end of the passenger strand of a non-segmented duplex miRNA mimetic is position 1 of passenger strand, the nucleotide immediately adjacent to it is position 2, and so on and so forth Likewise, the first nucleotide position at the 5′-end of the guide strand of the non-segmented duplex miRNA mimetic is position 1 of the guide strand, the nucleotide immediately adjacent to it is position 2, and so on and so forth.

By “a contiguous stretch of nucleotides” or “a contiguous stretch of nucleotide positions” is meant a continuous series of at least 2 nucleotides or at least two nucleotide positions. For example, a contiguous stretch of nucleotides can refer to an unsegmented or uninterrupted oligonucleotide of 2 to 20 nucleotides in length. When referring to a contiguous stretch of nucleotides, the bonds connecting the nucleotides within the stretch can be phosphodiester bonds or non-phosphodiester linkages. A gap comprising a contiguous stretch of nucleotide positions can refer to a gap occupying, for example, from 1 to 10 or more nucleotide positions.

A segmented miRNA mimetic of the invention provided to a cell is typically designed based on the sequence of a naturally-occurring miRNA in the cell. As such, the naturally-occurring miRNA in the cell is referred to herein as “the corresponding miRNA.” A segmented miRNA mimetic of the invention provided to a cell is also understood to target one or more target mRNAs that are also targeted by the corresponding naturally-occurring miRNA. As such, each RNA targeted by the corresponding naturally-occurring miRNA is referred to as “the corresponding miRNA target.” It is contemplated that a segmented miRNA molecule introduced to a cell is not necessarily or does not necessarily comprise a nucleic acid sequence that is identical, essentially homologous, or even substantially homologous to a naturally-occurring miRNA, but the segmented miRNA is capable of either becoming or functioning as a naturally-occurring miRNA under appropriate conditions.

The term “discontinuity” as used herein refers to a non-contiguous segment of the nucleotide sequence of the guide strand, passenger strand or both the passenger and guide strands of a segmented micro RNA mimetic. A discontinuity can include one or more nicks, gaps, substitutions or insertions. The discontinuity can comprise, for example, from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more unoccupied or vacant nucleotide positions in the guide strand, the passenger strand, or both the guide and passenger strands. For example, a nick will comprise 0 unoccupied or vacant nucleotide positions, whereas a gap will comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10 or more) vacant or unoccupied nucleotide positions. Likewise, the discontinuity can comprise, for example, from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more nucleotide positions that are occupied or replaced by a non-nucleotide moiety in the guide strand, the passenger strand, or both the guide and passenger strands. For example, an insertion will comprise a non-nucleotide moiety that can occupy 0 nucleotide positions, whereas a substitution will comprise a non-nucleotide moiety that occupies one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10 or more) otherwise vacant nucleotide positions.

As used herein, “endogenous” refers to its meaning as is generally accepted in the art. The term generally refers to any material from or produced inside an organism, cell, tissue or system. As used herein, an “endogenous miRNA” is a naturally-occurring miRNA in a cell, tissue, organism, including a mammal, such as, for example, a human. “Exogenous” generally refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term “expression” refers to its meaning as is generally accepted in the art. The term generally refers to the transcription and/or translation of a particular nucleotide sequence, for example when driven by its promoter.

The term “gap” as used herein refers to a contiguous stretch of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more), internal (as opposed to “terminal”) vacant, unoccupied or “unfilled” nucleotide positions in one or both strands of a segmented miRNA mimetic of the invention. The gap can be present in the guide strand, the passenger strand, or in both guide and passenger strands of a segmented microRNA mimetic of the invention.

The term “gene” as used herein, especially in the context of “target gene” for an RNAi agent, refers to the meaning as is generally accepted in the art. The term generally refers to a nucleic acid (e.g., “target DNA” or “target RNA”) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide. The target gene can also include a UTR (i.e., untranslated region) or non-coding region of the nucleic acid sequence. A gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA) as are generally known in the art, such as endogenous antisense RNA, small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof, or any other regulatory RNA or precursor thereof. Such non-coding RNAs can serve as target nucleic acid molecules for RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Aberrant fRNA or ncRNA activity leading to disease can therefore be modulated by the RNAi agents of the invention. RNAi agents targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.). A target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. A cell containing a target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts. For a review, see for example Snyder and Gerstein, 2003, Science 300:258-260. In one aspect of the present invention, a segmented miRNA mimetic is capable of exerting regulatory effects on multiple target genes. Also, at least one of these target genes, but typically more than one target genes, can be shared between a segmented miRNA mimetic of the invention and its corresponding endogenous miRNA.

As used herein, “gene silencing” refers to a partial or complete loss-of-function through targeted inhibition of an endogenous miRNA target in a cell. As such, the term is used interchangeably with RNAi, “knockdown,” “inhibition,” “down-regulation,” or “reduction” of expression of a miRNA target gene. Depending on the circumstances and biological problem to be addressed, it can sometimes be preferable to increase expression of one or more related genes, which is termed “up-regulation” herein. Alternatively, it might be desirable to reduce or increase gene expression as much as possible or only to a certain extent.

By “guide strand” of a segmented miRNA of the invention is meant two or more distinct contiguous stretches of nucleotides at least one of which is substantially homologous or identical to the whole or a part of a sequence of a corresponding naturally-occurring miRNA, such as one selected from the miRBase, and for example, such as one selected from SEQ ID NOs: 1-1090 of Table I herein. The nucleotides within each contiguous stretch can be connected by traditional phosphodiester bonds and/or non-phosphodiester connectors. In addition, the guide strand of a segmented miRNA mimetic can comprise two or more distinct stretches of nucleotides that are capable of forming base pairs with the nucleotides or residues at the complementary nucleotide positions of the passenger strand.

As used herein, the term “homologous” (or “homology”) refers to its meaning as is generally accepted in the art. The term generally refers to the number of nucleotides of the subject nucleic acid sequence that has been matched to identical nucleotides of a reference nucleic acid sequence, typically by a sequence analysis program or by visual inspection. For example, nucleic acid sequences can be compared using computer programs that align the similar sequences of nucleic acids and therefore define the differences. Exemplary computer programs includes the BLAST program (NCBI) and parameters used therein, as well as the DNAstar system (Madison, Wis.), which can be used to align sequence fragments. Equivalent alignments and assessments can also be obtained through the use of any standard alignment software.

As used herein, the terms “including” (and any form thereof, such as “includes” and “include), “comprising” (and any form thereof, such as “comprise” and “comprises”), “having” (and any form thereof, such as “has” or “have”), or “containing” (and any form thereof, such as “contains” or “contain”) are inclusive and open-ended and do not exclude additional, un-recited elements or method steps.

The term “insertion” as used herein refers to a discontinuity wherein one or more non-nucleotide moieties are incorporated into the guide strand and/or passenger strand, while preserving the base pairs in the guide and passenger strands. Examples of such non-nucleotide moieties are provided herein and others are provided as is generally known to those of skill in the art.

The term “internal ends” refers to the ultimate nucleotides of the contiguous stretches of nucleotides on either side of a gap or a nick. Gaps or nicks do not have “terminal ends” for the purpose of this disclosure.

As used herein, the term “internally unpaired nucleotides” refers to nucleotides, which do not form base pairs with nucleotides at the complementary nucleotide positions in the opposite strand according to the standard Waston-Crick base-pairing rules. The term “internally unpaired nucleotides” also refers to nucleotide analogs or non-nucleotide residues that do not form hydrogen bonds or base pairs with the nucleotides, nucleotide analogs, or non-nucleotide residues at the complementary nucleotide positions in the opposite strand.

In certain embodiments, a segmented miRNA of the invention can be isolated. As used herein, an “isolated” oligonucleotide is nucleic acid molecule that exists in a physical form differing from any nucleic acid molecules of identical sequence as found in nature. “Isolated” does not require, although it does not prohibit, that the nucleic acid be physically removed from its native environment. For example, a nucleic acid can be said to be “isolated” when it includes nucleotides and/or internucleotide bonds not found in nature. A nucleic acid can be said to be “isolated” when it exists at a purity not found in nature, where purity can be adjudged with respect to the presence of nucleic acids of other sequences, with respect to the presence of proteins, with respect to the presence of lipids, or with respect to the presence of any other component of a biological cell, or when the nucleic acid lacks sequence that flanks an otherwise identical sequence in an organism's genome, or when the nucleic acid possesses sequence not identically present in nature. In aspects of the invention, a segmented miRNA is isolated by virtue of its having been synthesized in vitro. It will be understood, however, that isolated nucleic acids can be subsequently mixed or pooled together.

As used herein, the term “locked nucleic acid” (LNA) refers to its meaning as is generally accepted in the art. The term generally refers to a structure of the general Formula I:

where X and Y are independently selected from the group consisting of —O—, —S—, —N(H)—, —N(R)—, —CH₂—, or —CH— (if part of a double bond), —CH₂—O—, CH₂—S—, CH₂—N(H)—, —CH₂—N(R)—, —CH₂—CH₂—, and CH₂—CH-(if part of a double bond), —CH═CH—, where R is selected from hydrogen and C₁₋₄-alkyl; Z and Z* are independently selected from an internucleotide linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleobase; and the asymmetric groups can be found in either orientation.

The 4 chiral centers of Formula I, as shown, are in a fixed configuration. But their configurations are not necessary fixed. Also comprised in the invention are compounds of the generally Formula I, wherein the chiral centers are found in different configurations, such as those represented in Formula II (below). Thus each chiral center in Formula I can exist in either R or S configuration. The definition of R (rectus) and S (sininster) are described in the IUPAC 1974 Recommendations, Section E, Fundamental Setereochemistry: The rules can be found in Pure Appl. Chem. 45, 13-30 (1976) and In “Nomenclature of Organic Chemistry” pergamon, New York, 1979.

LNA compounds can include an activation group for —OH, —SH, and —NH(R^(H)), respectively. Such activation groups are, for example, selected from optionally substituted O-phosphoramidite, optionally substituted O-phosphortriester, optionally substituted O-phosphordiester, optionally substituted H-phosphonate, and optionally substituted O-phosphonate.

B constitutes a natural or non-natural nucleobase and selected among adenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine, guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil, 5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine, and 2-chloro-6-aminopurine.

Preferably, the Locked Nucleic Acid (LNA) used in a segmented miRNA mimetic of the invention comprises at least one nucleotide comprises a Locked Nucleic Acid (LNA) unit according any of the Formulas II:

wherein Y is —O—, —S—, —NH—, or N(R^(H)); Z and Z* are independently selected among an internucleotide linkage, a terminal group or a protecting group; and B constitutes a natural or non-natural nucleobase. These exemplary LNA monomers and others, as well as their preparation are described in WO 99/14226 and subsequent applications, WO 00/56746, WO 00/56748, WO 00/66604, WO 00/125248, WO 02/28875, WO 2002/094250 and WO 2003/006475, the disclosure of all of which are incorporated herein by reference.

As used herein, the term “mimetic” refers to its meaning as is generally accepted in the art. The term generally refers to a molecule that is structurally different from the reference molecule (e.g., the corresponding naturally-existing molecule or the corresponding non-segmented mimetic molecule) but is capable of performing one or more or all of the biological, physiological, and/or chemical functions that are within the capabilities of the references molecule. The mimetic and the reference molecule do not have to be functional equivalents but the mimetic should be able to perform one or more functions, and exhibiting at least 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of the reference molecule, as measured and compared using assays or parameters that are suitable to represent the shared function(s). As used herein, a segmented miRNA molecule is a miRNA mimetic when the former shares at least one function with its corresponding endogenous miRNA. A miRNA mimetic can be a synthetic RNA duplex, such as a segmented miRNA duplex of the invention, a vector-encoded hairpin molecule, or other suitable structures designed based on a corresponding naturally-occurring endogenous miRNA.

The term, “miRNA” or “microRNA” refers to its meaning as is generally accepted in the art. The term generally refers to an endogenous short RNA molecule, which can be isolated or synthetic, which is found in eukaryotes and is involved in RNA-based gene regulation. A representative set of known endogenous miRNA species is described in the publicly available miRBase sequence database as described in Griffith-Jones et al., Nucleic Acids Research, 2004, 32:D109-D111 and Griffith-Jones et al., Nucleic Acids Research, 2006, 34:D 140-D144, accessible on the World Wide Web at the Wellcome Trust Sanger Institute website. A more selected set of miRNA species are included in Table I herein. Each mature miRNA is partially complementary to one or more messenger RNA (mRNA) molecules, which are also called “miRNA targets,” thereby regulating the expression of genes associated with the miRNA targets.

The term “nick” as used herein refers to a break in an internucleotide linkage in one or both strands of a segmented miRNA mimetic of the invention.

The term “non-nucleotide” refers to its meaning as is generally accepted in the art. The term generally refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, such as for example but not limitation abasic moieties, alkyl moieties, polymers such as PEG, peptides, sterols, peptide nucleic acids, and the like.

The term “nucleotide” refers to its meaning as is generally accepted in the art. The term generally refers to compounds that comprise a nucleobase, a sugar, and an internucleoside linkage, e.g., a phosphate group such as a phosphodiester. The base can be a natural bases (standard), modified bases, or a base analog, as are well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Additionally, the nucleotides can be unmodified or modified at the sugar, internucleoside linkage, and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others; see, for example, U.S. application Ser. No. 12/064,014).

The term “parenteral,” refers to its meaning as is generally accepted in the art. The term generally includes subcutaneous, intravenous, intramuscular, intraarterial, intraabdominal, intraperitoneal, intraarticular, intraocular or retrobulbar, intraaural, intrathecal, intracavitary, intracelial, intraspinal, intrapulmonary or transpulmonary, intrasynovial, and intraurethral injection or infusion techniques.

By “passenger strand” of a segmented miRNA of the invention is meant one or more distinct nucleic acid sequences or contiguous stretches of nucleotides capable of forming base pairs (including traditional base pairs and non-traditional base pairs) to one or more non-overlapping contiguous stretches of nucleotides in the guide strand. The nucleotides within each contiguous stretch can be connected by traditional phosphodiester bonds and/or non-phosphodiester connectors. In addition, the passenger strand of a segmented miRNA can comprise one or more nucleic acid sequences having at least substantial homology, or at least essential homology, or even perfect homology to a RNA sequence that is a target of a corresponding naturally-occurring miRNA, such as one selected from the miRBase, and for example, one selected from Table I herein.

The terms “patient,” “subject,” “individual” refer to their ordinary meanings as are generally accepted in the art. The terms generally refer to any animal or cells or tissues thereof whether in vitro or in situ, amendable to the methods described herein. They typically refer to an organism, which is a donor or recipient of explanted cells or the cells themselves. They also refer to an organism to which the segmented miRNAs of this disclosure can be administered. In certain non-limiting embodiments, the patient, subject or individual is a mammal or a mammalian cell. In other non-limiting embodiments, the patient, subject or individual is a human or a human cell.

The term “phospholipid” refers to its meaning as is generally accepted in the art. The term generally refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

The term “perfect complementarity” (or “perfectly complement”) as used herein refers to complete (100%) complementarity within a contiguous region of double-stranded nucleic acid, such as, for example, between a hexamer or heptamer seed sequence of a miRNA and its complementary sequence in a target mRNA. “Perfectly complementary” can also mean that all the contiguous residues of a first nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. For example, 2 or more perfectly complementary nucleic acid strands can have the same number of nucleotides (i.e., have the same length and form one double-stranded region with or without an overhang), or have a different number of nucleotides (e.g., one strand can be shorter but fully contained within a second strand). “Perfect complements” can be formed between modified nucleotides and nucleotide analogs.

The term “perfect homology” (or “perfectly homologous”) as used herein refers to complete (100%) homology or “identity” between a reference sequence and a subject nucleic acid sequence. When there is a perfect homology, the reference and the subject sequences are the same.

The term “phosphorothioate” refers to its meaning as is generally accepted in the art. The term generally refers a sulphur substituted internucleotide phosphate linkage, but can also refer to internucleotide linkages selected from the group consisting of: —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —OP(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—, —O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—, and/or the internucleotide linkage can be selected form the group consisting of: —O—CO—O—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—, —O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—, —CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—, where R^(H) is selected from hydrogen and C₁₋₄-alkyl, Suitably, in some embodiments, sulphur (S) containing internucleotide linkages as provided above can be preferred. Moreover, a segmented miRNA mimetic of the invention can comprise other linkages or mixtures of different linkages—for example, both phosphate or phosphorothioate linkages, or just phosphate linkages, or other linkages as described herein.

The terms “polynucleotide” and “oligonucleotide” refer to their meanings as are generally accepted in the art. The terms generally refers to a chain of nucleotides. “Nucleic acids” or “nucleic acid molecules” are polymers of nucleotides. Thus, “nucleic acids” and “polynucleotides” or “oligonucleotides” are interchangeable herein. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric nucleotides. The monomeric nucleotides can be further hydrolyzed into nucleosides.

The term “protecting group” refers to its meaning as is generally accepted in the art. Protection groups of hydroxy substituents comprises substituted trityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy (MMT), and trityloxy, optionally substituted 9-(9-phenyl)xanthenyloxy (pixyl), optionally substituted methoxytetrahydro-pyranyloxy (mthp), silyloxy such as trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS) tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy, and phenyldimethylsilyloxy, tert-butylethers, acetals (including two hydroxy groups), acyloxy such as acetyl or halogen substituted acetyls.

The term “purine” refers to its meaning as is generally accepted in the art. The term generally refers to conventional purine nucleotides, including those with standard purine bases adenine and guanine. In addition, the term “purine” is contemplated to embrace nucleotides with natural non-standard purine bases or acids, such as N₂-methylguanine, inosine, 2,6-diaminopurine and the like, as well as chemically modified bases or “universal bases,” which can be used to substitute for standard urines herein.

The term “pyrimidine” refers to its meaning as is generally accepted in the art. The term generally refers to conventional pyrimidine nucleotides, including those with standard pyrimidine bases uracil, thymidine and cytosine. In addition, the term pyrimidine is contemplated to embrace nucleotides with natural non-standard pyrimidine bases or acids, such as 5-methyluracil, 2-thio-5-methyluracil, 4-thiouracil, pseudouracil, dihydrouracil, orotate, 5-methylcytosine, or the like, as well as a chemically modified bases or “universal bases,” which can be used to substitute for a standard pyrimidine within the nucleic acid molecules of this disclosure.

The term “RNA” refers to its meaning as is generally accepted in the art. The term generally refers to a molecule comprising at least one ribofuranoside residue, such as a ribonucleotide. The term “ribonucleotide” means a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety. The term refers to a double-stranded RNA, a single-stranded RNA, an isolated RNA such as a partially purified RNA, an essentially pure RNA, a synthetic RNA, a recombinantly produced RNA, or an altered RNA that differs from a naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides therein. Such alterations can include addition of non-nucleotide material, for example, at one or more non-terminal nucleotides of an RNA molecule. As such, nucleotides in the segmented miRNA mimetics of the invention can comprise non-standard nucleotides, such as non-naturally occurring nucleotides, chemically synthesized and/or modified nucleotides, or deoxynucleotides. The altered RNAs are referred to as “RNA analogs” or “analogs of naturally-occurring RNA” containing standard nucleotides (i.e., adenine, cytidine, guanidine, thymidine and uridine), or generally as “modified RNA”.

As used herein, the phrase “RNA interference” (also called “RNAi” herein) refers to its meaning as is generally accepted in the art. The term generally refers to the biological process of inhibiting, decreasing, or down-regulating gene expression in a cell, and which is mediated by short interfering nucleic acid molecules (e.g., siRNAs, miRNAs, shRNAs), see for example Zamore and Haley, 2005, Science 309:1519-1524; Vaughn and Martienssen, 2005, Science 309:1525-1526; Zamore et al., 2000, Cell 101:25-33; Bass, 2001, Nature 411:428-429; Elbashir et al., 2001, Nature 411:494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hall et al., 2002, Science 297:2232-2237; Hutvagner and Zamore, 2002, Science 297:2056-60; McManus et al., 2002, RNA 8:842-850; Reinhart et al., 2002, Gene & Dev. 16:1616-1626; and Reinhart & Bartel, 2002, Science 297:1831). Additionally, the term “RNA interference” (or “RNAi”) is meant to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetics. For example, segmented microRNA mimetics of the invention can be used to epigenetically silence genes at either the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by segmented microRNA mimetics of the invention can result from modification of chromatin structure or methylation patterns to alter gene expression (see, for example, Verdel et al., 2004, Science 303:672-676; Pal-Bhadra et al., 2004, Science 303:669-672; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hall et al., 2002, Science 297:2232-2237). In another non-limiting example, modulation of gene expression by segmented microRNA mimetics of the invention can result from cleavage of RNA (either coding or non-coding RNA) via RISC, or via translational inhibition, as is known in the art or modulation can result from transcriptional inhibition (see for example Janowski et al., 2005, Nature Chemical Biology 1:216-222).

The term “RNA profile” or “gene expression profile” refers to a set of data regarding the expression pattern for one or more gene or genetic marker in the sample (e.g., a plurality of nucleic acid probes that identify one or more markers). In some embodiments, it can be useful to know whether a cell expresses a particular miRNA endogenously or whether such expression is affected under particular conditions or when it is in a particular disease state. Thus in some embodiments of the invention, methods include assaying a cell or a sample containing a cell for the presence of one or more marker genes or mRNA or other analyte indicative of the expression level of a gene of interest. Consequently in some embodiments, methods include a step of generating an RNA profile for a sample.

As used herein, the term “seed sequence” refers to at least 6 consecutive nucleotides within any of nucleotide positions 1 to 10 of the 5′-end of a naturally-occurring mature miRNA, such as one selected from those listed in miRBase (http://www.mirbase.org/) as of the filing date of the present application, and for example, such as one selected from those listed in Table I, wherein the seed sequence nucleotides of positions 1 to 8 are capitalized. See, e.g., Brennecke et al., 2005, PLOS Biol. 3(3):e85. In a naturally-occurring miRNA, the seed sequence typically determines the target mRNA sequence to which the miRNA can bind and provide gene regulation. As such, multiple naturally-occurring miRNAs can share a seed sequence, or share substantial homology in the seed sequences, and these miRNAs are members of the same miRNA family.

The term “segmented miRNA mimetic” (or “segmented miRNA,” interchangeably) as used herein refers to a miRNA mimetic molecule comprising at least one discontinuity in the guide strand that is capable of modulating the expression of a target gene that is also regulated by a corresponding naturally-occurring miRNA, such as one selected from the miRBase as of the filing date of the present application, and for example, such as one selected from SEQ ID NOs: 1-1090 of Table I herein. The discontinuity comprises one or more nicks, gaps, substitutions, or insertions. In one aspect, a segmented miRNA mimetic of the invention will mediate gene silencing via an RNAi mechanism such as RISC mediated cleavage, translational inhibition, or epigenetic silencing as is known in the art. A segmented miRNA of the invention can comprise one or more or all ribonucleotides. Segmented miRNAs of the invention can also comprise nucleotide and non-nucleotide analogs as described herein and as otherwise known in the art.

A segmented miRNA mimetic of the invention is said to be “double-stranded” if the molecule has an overall double-stranded conformation. Each of the “strands” is not necessarily continuous, but rather can comprise one or more distinct contiguous stretches of nucleotides, separated by non-contiguous segments (i.e. gaps, nicks, substitutions, insertions). The strand (including the one or more contiguous stretches of nucleotides) that comprises or comprises essentially of a sequence of a corresponding miRNA target is termed the “passenger strand of the segmented miRNA.” The strand (including the one or more contiguous stretches of nucleotides) that comprises or comprises essentially of at least a portion (e.g., a stretch of about 5 to about 8 nucleotides within the seed sequence) of a corresponding endogenous miRNA is termed the “guide strand of the segmented miRNA.” Moreover, a guide strand comprising one or more discontinuities (i.e., gaps, nicks, substitutions, insertions) can form a double-stranded RNA complex even if it is hybridized to a passenger strand that also comprises one or more discontinuities (i.e., gaps, nicks, substitutions, insertions). When both strands comprise discontinuities, the discontinuities can, in certain embodiments, be arranged in such relative positions with each other that the segmented miRNA mimetic of the invention maintains a generally double-stranded conformation, thereby allowing its recognition by the cellular RNAi machinery. Linkers can be introduced and various other stabilizing modifications can be applied to confer added thermodynamic stability. In a specific embodiment, linkers or stabilizing modifications are introduced to a molecule comprising structurally overlapping discontinuities, where otherwise the double-stranded molecule would have broken into two double-stranded sections if the contiguous stretches adjacent to the overlapping gaps are not connected.

Each segmented miRNA mimetic of the invention can have a corresponding non-segmented double-stranded miRNA mimetic, where the non-segmented mimetic comprises all of the contiguous stretches of nucleotides of the segmented mimetic, and where the non-segmented mimetic and the segmented mimetic share the same RNA target(s) with their corresponding endogenous miRNA. Essentially, the segmented miRNA mimetic is designed based on the corresponding non-segmented miRNA mimetic by deleting certain internal phosphodiester backbone linkages and/or internal nucleotides (i.e., by placing nicks or gaps) or substituting such nicks or gaps with one or more non-nucleotide moieties.

It is contemplated that multiple segmented miRNAs, having respective multiple corresponding miRNAs, can be applied to a cell. In particular embodiments, two or more segmented miRNAs are introduced to a cell. A combination of multiple segmented miRNAs can act as one or more points of regulation in cellular pathways within the cell, which has aberrant phenotype(s) (i.e., that the cell is a “targeted cell”), and that such combination can have increased efficacy for correcting the aberrant phenotype(s) of the targeted cell. If the targeted cell is mixed with normal cells, it is contemplated that the segmented miRNAs added to the collection of cells, while providing improved efficacy to correct the aberrant phenotypes of the targeted cell, have minimal adverse effect on the normal cells.

The term “siRNA” (also “short interfering RNA” or “small interfering RNA”) is given its ordinary meaning as is recognized in the art.

A double-stranded nucleic acid molecule can have strands that are not perfectly complementary, but merely “substantially complementary.” By “substantially complementary” it is meant that the nucleic acid sequence of the first strand is at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the nucleic acid sequence of the second strand. In certain embodiments, complementary nucleic acid molecules can have wrongly paired bases—that is, bases that cannot form a traditional Waston-Crick base pair (i.e., forming a hydrogen bond) or other non-traditional types of base pair (i.e., “mismatched” bases, formed or held together by non-traditional forces that are not hydrogen bonds). Non-traditional Waston-Crick base pairs are also understood as “non-canonical base pairs,” which is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+ carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

As used herein, the term “substantially homologous” (or “substantial homology”) is meant that the subject sequence shares at least 25% (e.g., at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) homologous nucleotides with the nucleotides of the same nucleotide positions in a reference sequence. By “essentially homologous” (or “essential homology’) it is meant that, a first part of a subject sequence having a number of consecutive nucleotides is identical to a first part of a reference sequence having the same number or consecutive nucleotides, whereas the rest of the subject sequence, which does not overlap with the first part of the subject sequence, is substantially homologous to the rest of the reference sequence, which does not overlap with the first part of the reference sequence. For example, as used herein, the term “essentially homologous” with regard to miRNA sequences, can refer to the contiguous stretch from the 5′-terminal of the guide strand of a segmented miRNA mimetic of the invention comprising a sequence that is essentially homologous to a sequence, including the seed sequence, of a corresponding naturally-occurring miRNA. For example, the first contiguous stretch from the 5′-terminal of the guide strand can comprise a 6 to 7-nucleotide stretch within that is perfectly complementary to a 6 to 7-nucleotide stretch of the seed sequence, where the rest of the nucleotides (including nucleotide analogs) in the contiguous stretch can be at least 50% homologous to the rest of the corresponding endogenous mature miRNA sequence. The comparison of sequences and determination of percent homology and/or identity between two sequences can be accomplished using a mathematical algorithm of Karlin and Altschul (1990, PNAS 87:2264-2268), modified as in Karlin and Altschul (1993, PNAS 90:5873-5877) or by visual inspection.

As used herein, the term “substitute non-nucleotide moieties” refers to chemical moieties that are capable of substituting one or more nucleotides in a segmented miRNA mimetic of the invention. The substitute non-nucleotide moieties can allow for non-traditional base-pairing (i.e., not forming traditional hydrogen bonds) between the strands and contribute to the binding free energy. In certain embodiments, the substitute non-nucleotide moieties of the instant disclosure are those that are capable of associating or otherwise interacting with one or more components of the cellular RNAi machinery, including, for example, the PAZ domain, the PIWI domain and/or other Argonaute protein domains associated with the RISC.

The term “substitution” as used herein refers to a discontinuity in which one or more nucleotide(s) of the otherwise continuous nucleotide sequence of the guide strand and/or passenger strand is replaced with one or more non-nucleotide moieties. Examples of such non-nucleotide moieties are provided herein and others are provided as is generally known to those of skill in the art.

The segmented miRNAs of the invention are typically synthetic. The term “synthetic” as used herein generally refers to nucleic acid molecules that are not produced naturally in a cell. In certain aspects, the chemical structure of a synthetic nucleic acid molecule can deviate from a naturally-occurring nucleic acid molecule. On the other hand, a synthesized segmented miRNA can encompass all or part of a naturally-occurring miRNA sequence or a component thereof. Moreover, it is contemplated that, in a specific embodiment, a synthetic segmented miRNA mimetic administered to a cell can subsequently be altered or processed by the cellular components such that its post-processing structure or sequence can be identical to the whole or a part of a naturally-occurring miRNA. The difference between a synthetic miRNA mimetic and its corresponding endogenous miRNA, including miRNA precursors and complements, can comprise missing internal (i.e., at non-terminal positions) phosphordiester bonds, or missing internal nucleotides, altered types of nucleotides, altered internucleotide connectors or linkages, or chemically modified nucleotides. In certain aspects, a synthetic segmented miRNA of the invention is an RNA or an RNA analog.

The phrases “target site,” “target sequence,” and “target region,” as used herein, refer to their meanings as generally accepted in the art. The terms generally refer to a sequence within a target nucleic acid molecule (e.g., target RNA) that is “targeted,” e.g., for cleavage mediated by an RNAi molecule that contains a sequence within its guide/antisense region that is partially, substantially, or perfectly complementary to that target sequence. A “target site” for a miRNA mimetic molecule of the present invention refers to a nucleic acid sequence that is partially, substantially, or perfectly complementary to the guide strand of the miRNA mimetic. The target site can be within a coding or a non-coding (i.e., untranslated) region of a target RNA. The target site can be the target site for an endogenous miRNA for which the segmented miRNA molecule is a mimetic, in which case the “target site” can also be referred to as a “miRNA target site” or a “corresponding miRNA target site.”

Linkers connecting the terminal ends of a segmented miRNA mimetic of the invention are referred to as “terminal linkers” herein.

The term “therapeutic” refers to its meaning as is generally accepted in the art. The term generally refers to a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state. In the instant application, the disease state is particularly referred to as one associated with aberrant biological pathways regulated by miRNAs, such as those listed in the miRBase at the time of filing of this application, and especially those listed in Table I herein. The term “treatment” as used herein is meant to include therapeutic treatment as well as prophylactic, or suppressive measures for diseases or disorders. Thus, for example, the term “treatment” includes the administration of an agent prior to or following the onset of a disease or disorder thereby preventing or removing all signs of the disease or disorder. As another example, administration of the agent after clinical manifestation of the disease to combat the symptoms of the diseases is also comprised by the term “treatment.”

As used herein, the term “therapeutically effective amount” refers to its meaning as is generally accepted in the art. The term can refer to an amount of a segmented miRNA that is sufficient to result in a decrease in severity of disease symptoms, an increase in frequency or duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease, in the subject (e.g., a mammal or human) to which it is administered. One of ordinary skill in the art would be able to determine such therapeutically effective amounts based on such factors such as the subject's size, the severity of symptoms, and the particular composition or route of administration selected. For example, a therapeutically effective amount of a segmented miRNA of the invention, individually, in combination, or in conjunction with other drugs, can be used or administered at a therapeutically effective amount to a subject or by administering to a particular cells under conditions suitable for treatment, to, for example, decrease tumor size, or otherwise ameliorate symptoms associated with a particular disorder in the subject.

As used herein, “terminals” or “terminal ends” refers to the ultimate ends at the first 5′-nucleotide or the first 3′-nucleotide of a given strand. Substitutions of such terminal ends can be selected independently from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—, Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionally substituted C₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl, optionally substituted C₂₋₆-alkynyloxy, monophosphate, monothiophosphate, diphosphate, dithiophosphate triphosphate, trithiophosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—, aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—, carboxy methyl, sulphonomethyl, where Prot is a protection group for —OH, —SH, and —NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and —NH(R^(H)), respectively, and R^(H) is selected from hydrogen and C₁₋₆-alkyl.

Linkers connecting the terminal ends of a segmented miRNA mimetic of the invention are referred to as “terminal linkers” herein.

The term “universal base” refers to its meaning as is generally accepted in the art. The term generally refers to nucleotide base analogs that form base pairs with each of the standard DNA/RNA bases with little discrimination among them, and is recognized by intracellular enzymes. See, e.g., Loakes et al., J. Mol. Bio. 1997, 270:426-435. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carbozamides, and nitroazole derivatives such as 3′-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art. See, e.g., Loakes, 2001 Nucleic Acids Res. 29:2437.

A “vector” refers to its meaning as is generally accepted in the art. The term generally refers to a replicon, such as a plasmid, phagemid, cosmid, baculovirus, bacmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), as well as other bacterial, yeast, or viral vectors, to which another nucleic acid segment can be operatively inserted so as to bring about replication or expression of the inserted segment. “Expression vector” refers to a vector comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

Any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range, and when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

Other objects, features and advantages of the present invention will become apparent from the detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.

A Segmented miRNA of the Invention

The instant disclosure provides a segmented miRNA mimetic molecule (segmented miRNA mimetic) that is double-stranded comprising a passenger strand and a distinct guide strand, wherein at least the guide strand includes one of more discontinuities and wherein the passenger strand and the guide strand each independently comprises, in sum, about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides, and the mimetic molecule comprises, in sum, about 10 to about 26 (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) base pairs. In one aspect, a segmented miRNA of the invention comprises 2 or more (e.g., 2, 3, or 4) distinct contiguous stretches of nucleotides in the guide strand. A “contiguous stretch of nucleotides” can comprise as little as 2 nucleotides, according to the present invention. In another aspect, a segmented miRNA of the invention comprises 2 or more (e.g., from 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more distinct contiguous stretches of nucleotides in each of the passenger strand and the guide strand. The distinct contiguous stretches of nucleotides are arranged such that their complementary sequences located on the opposite strand or within a corresponding miRNA target sequence do not overlap. Within each of the contiguous stretches of nucleotides, the nucleotides are connected by phosphodiester bonds and/or non-phosphodiester connectors. The distinct contiguous stretches of nucleotides in a given strand are arranged from 5′- to 3′-, and the each pair of neighboring stretches can be separated by a nick, gap, substitution, or insertion.

In one embodiment, a first discontinuity in the passenger strand and a second discontinuity in the guide strand of a segmented miRNA mimetic do not overlap provided that RNAi activity against one or more miRNA targets is maintained.

In another embodiment, a first discontinuity in the passenger strand and a second discontinuity in the guide strand of a segmented miRNA mimetic partially overlap provided that RNAi activity against one or more miRNA targets is maintained.

In another embodiment, a first discontinuity in the passenger strand and a second discontinuity in the guide strand of a segmented miRNA mimetic overlap completely provided that RNAi activity against one or more miRNA targets is maintained. For example, overlapping nicks or gaps can result in a miRNA mimetic molecule that is no longer able to associate into duplex form. One of skill in the art will readily appreciate that such designs are to be avoided in order to maintain miRNA medicated RNAi activity.

In one embodiment, a first nick in the passenger strand and a second nick in the guide strand of a segmented miRNA mimetic do not overlap.

In one embodiment, a first gap in the passenger strand and a second gap in the guide strand of a segmented miRNA mimetic do not overlap by at least one complementary nucleotide position.

In one aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula III:

wherein the molecule comprises a passenger strand and a guide strand, where each line shown in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2” and “X3” represent the number of nucleotide positions in each stretch, “G/N” represents a discontinuity in the guide strand, “Y1” represents a number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides.

With reference to Formula III, in one embodiment, X1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X2 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X3 is an integer from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X2, X3 and Y1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide strand is a nick. In certain embodiments the discontinuity in the guide strand is a gap. In certain embodiments the discontinuity in the guide strand is a substitution. In certain embodiments the discontinuity in the guide strand is an insertion. In one embodiment, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, i.e., wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In a further embodiment, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strand, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

With reference to Formula III, in one embodiment, X1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X2 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X3 is an integer from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X2, X3 and Y1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide strand is a nick. In certain embodiments the discontinuity in the guide strand is a gap. In certain embodiments the discontinuity in the guide strand is a substitution. In certain embodiments the discontinuity in the guide strand is an insertion. In one embodiment, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, i.e., wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In a further embodiment, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strand, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

In another aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula IV:

wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3” and “X4” represents a number of nucleotide positions in each stretch, “P/N” represents a discontinuity in the passenger strand, “G/N” represents a discontinuity in the guide strand, “P/N” represents a discontinuity in the passenger strand, each of “Y1” and “Y2” represents a number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides.

With reference to Formula IV, in one embodiment, X1 and X2 are integers independently from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2 and Y1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X3 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X4 is an integer from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y2 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X3, X4 and Y2 is about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, i.e. wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

With reference to Formula IV, in one embodiment, X1 and X2 are integers independently from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2 and Y1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X3 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X4 is an integer from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y2 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X3, X4 and Y2 is about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, i.e. wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

In yet another aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula V:

wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3” and “X4” represents the number of nucleotide positions in each stretch, each “G/N” represents a discontinuity in the guide strand, each of “Y1” and “Y2” represents the number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides; wherein X1 is an integer from about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X2 and X3 are each independently an integer from about 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17), X4 is an integer from about 6 to about 22 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23), Y1 and Y2 are each independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X2, X3, X4, Y1 and Y2 is an integer from about 10 to about 26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

In a further aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula VI:

wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3,” “X4” and “X5” represents the number of nucleotide positions in each stretch, “P/N” represents a discontinuity in the passenger strand, “G/N” represents a discontinuity in the guide strand, each “P/N” represents a discontinuity in the passenger strand, each of “Y1,” “Y2” and “Y3” represents the number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides; wherein X1, X2, and X3 are each independently an integer from about 2 to about 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23), Y1 and Y2 are each independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2, X3, Y1 and Y2 is an integer from about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X4 is an integer from about 1 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X5 is an integer from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y3 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X4, X5 and Y3 is an integer from about 10 to about 26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

In a further aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula VII:

wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3,” “X4,” “X5” and “X6” represents the number of nucleotide positions in each stretch, each “P/N” represents a discontinuity in the passenger strand, each “G/N” represents a discontinuity in the guide strand, each of “Y1,” “Y2,” “Y3” and “Y4” represents the number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides; wherein X1, X2, and X3 are each independently an integer from about 2 to about 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23), Y1 and Y2 are each independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2, X3, Y1 and Y2 is an integer from about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X4 and X5 are each independently an integer from about 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17), X6 is an integer from about 7 to about 22 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23), Y3 and Y4 are independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X4, X5, X6, Y3 and Y4 is about 10 to about 26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

In a further aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula VIII:

wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3” and “X4” represents the number of nucleotide positions in each stretch, “P/N” represents a discontinuity in the passenger strand, each “G/N” represents a discontinuity in the guide strand, “P/N” represents a discontinuity in the passenger strand, each of “Y1” and “Y2” represents the number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides; wherein X1 and X2 are each independently an integer from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2 and Y1 is an integer from about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X3 and X4 are each independently an integer from about 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17), X5 is an integer from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y2 and Y3 are each independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X3, X4, X5, Y2 and Y3 is an integer from about 10 to about 26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

At least one of the 2 or more contiguous stretches of nucleotides in the guide strand of a segmented miRNA mimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises a sequence that is substantially, essentially or perfectly homologous (e.g., at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% homologous) to a nucleotide sequence of a naturally-occurring endogenous miRNA, such as one selected from miRBase as of the filing date of the present invention, and for example, one selected from SEQ ID NOs: 1-1090 of Table I herein. In certain embodiments, the first contiguous stretch of nucleotides from the 5′-end of the guide strand comprises at least 6 (e.g., 6, 7, or 8) consecutive nucleotides that are identical (or perfectly homologous) to a 6, 7, or 8-nucleotide sequence within the seed sequence of a naturally-occurring miRNA, such as one selected from Table I (wherein the seed sequence nucleotides are capitalized). In another embodiment, at least 2 of the 2 or more contiguous stretches of nucleotides in the guide strand of a segmented miRNA mimetic of Formula III-VIII comprise sequences that are substantially, essentially, or perfectly homologous (at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% homologous) to non-overlapping regions of a naturally-occurring endogenous miRNA. In yet another embodiment, all of the contiguous stretches of nucleotides in the guide strand comprise sequences that are substantially, essentially, or perfectly homologous (e.g., at least at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% homologous) to non-overlapping regions of a naturally-occurring endogenous miRNA. Each of the 1 or more contiguous stretches of nucleotides in the passenger strand of a segmented miRNA mimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises a sequence that is substantially or perfectly complementary (e.g., at least 25, 20, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% complementary) to a non-overlapping region of a naturally-occurring endogenous miRNA, such as one selected from miRBase as of the filing date of the present invention, and for example, one selected from SEQ ID NOs: 1-1090 of Table I herein.

At least one of the 2 or more contiguous stretches of nucleotides in the guide strand of a segmented miRNA mimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises sequence having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) contiguous nucleotides of a naturally-occurring endogenous miRNA, such as one selected from miRBase as of the filing date of the present invention, and for example, one selected from SEQ ID NOs: 1-1090 of Table I herein. In certain embodiments, the first contiguous stretch of nucleotides from the 5′-end of the guide strand comprises at least 6 (e.g., 6, 7, or 8) consecutive nucleotides of a seed sequence of a naturally-occurring miRNA, such as one selected from Table I (wherein the seed sequence nucleotides are capitalized). In another embodiment, at least 2 of the 2 or more contiguous stretches of nucleotides in the guide strand of a segmented miRNA mimetic of Formula III-VIII comprise sequence having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) contiguous nucleotides non-overlapping regions of a naturally-occurring endogenous miRNA. In yet another embodiment, all of the contiguous stretches of nucleotides in the guide strand comprises sequence having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) contiguous nucleotides to non-overlapping regions of a naturally-occurring endogenous miRNA. Each of the 1 or more contiguous stretches of nucleotides in the passenger strand of a segmented miRNA mimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises a sequence capable of forming 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26) base pairs to a non-overlapping region of a naturally-occurring endogenous miRNA, such as one selected from miRBase as of the filing date of the present invention, and for example, one selected from SEQ ID NOs: 1-1090 of Table I herein.

In one aspect, a segmented miRNA mimetic of the invention comprises two separate strands, a guide strand and a separate passenger strand, wherein the strands are not connected to each other at either the 5′ or the 3′ terminal ends by a linker. Linkers connecting the terminal ends of a segmented miRNA mimetic of the invention are referred to as “terminal linkers” herein. In another aspect, one or both terminal ends of a segmented miRNA mimetic molecule can be connected or linked together by a terminal nucleotide and/or non-nucleotide linker. In certain embodiments, either or both ends of the passenger strand and the guide strand of a segmented miRNA mimetic of the invention can be covalently linked by a terminal nucleotide and/or non-nucleotide linker as described herein and known in the art.

One or more substitutions or insertions can be present in the absence of any terminal linkers, as described above. Alternatively, one or more substitutions or insertions can be present in a given molecule with one or more terminal linkers.

One or more or all of nucleotides of each of the contiguous stretches of nucleotides can be ribonucleotides, modified ribonucleotides, or suitable nucleotide analogs. Incorporation of nucleotide analogs, such as various known sugar, base, and backbone modifications, and LNA monomer units into disrupted strands can significantly enhance serum stability and prolong target knockdown or expression regulatory effects. The segmented miRNA mimetic molecules of the present invention can functionally accommodate and are compatible with various chemical modifications, in various combinations and juxtapositions, and to varying degrees. For example, from one to all (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52) of the ribonucleotides of the segmented miRNA mimetics of the invention can be modified. The improved properties conferred by the functionally compatible chemical modifications to the sugar, base and/or backbone, or by including suitable nucleotide analog residues, are of particular importance for application of these segmented miRNA mimetic molecules in vivo, for example, for use as a therapeutic agent or as a functional genomic tool.

In certain embodiments, a segmented miRNA mimetic molecule of the present invention can comprise a 3′-terminal overhang in its passenger strand, guide strand, or both the passenger and guide strands. The “overhang” nucleotides are unpaired and single stranded regions located at the terminal ends of an otherwise generally double-stranded nucleic acid molecule. An exemplary segmented miRNA mimetic of the invention can comprise a 3′-terminal overhang of 1 to 5 nucleotides (e.g., 1, 2, 3, 4, or 5 nucleotides) in the passenger strand, the guide strand, or both the passenger and the guide strands. In alternative embodiments, a segmented miRNA mimetic of the present invention can be blunt-ended (i.e., comprising no terminal overhang nucleotides) at either or both terminal ends.

In a further aspect, the segmented miRNA mimetics of the invention, according to any of the embodiments herein, are capable of participating in RNAi against endogenous RNA targets of their corresponding naturally-occurring miRNAs. The inhibition of the miRNA target can be achieved via the standard miRNA-specific interference mechanism. For example, the inhibition of the miRNA target can be by interaction (e.g., base-paring, binding, etc.) with the untranslated mRNA region, with which the corresponding endogenous miRNA interacts, which effectuates the translational regulation of one or more downstream genes. Or, the inhibition of the miRNA target can be achieved via an siRNA-like interference mechanism wherein the binding of the miRNA target by the guide strand of the segmented miRNA mimetic results in the cleavage of the untranslated miRNA target.

Modified Segmented miRNA Mimetics

The introduction of modified nucleotide analogs into segmented miRNA mimetic molecules of the invention provides a tool for overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules (i.e., having standard nucleotides) that are exogenously delivered. In certain embodiments, the use of modified segmented miRNA mimetics of this disclosure can enable achievement of a given therapeutic effect at a lower dose since these molecules can be designed to have an increased melting temperature or half-life in a subject or biological samples (e.g., serum). Furthermore, certain modifications can be used to improve the bioavailability of segmented miRNA mimetics by targeting particular cells or tissues or improving cellular uptake of the segmented miRNA mimetics. Therefore, even if the activity of a segmented miRNA mimetic of this disclosure is somewhat reduced (e.g., by less than about 20%, or 30%, or even 40%) as compared to an unmodified segmented miRNA mimetic of the same structure, the overall activity of the modified segmented miRNA mimetic can be greater than that of its native counterpart due to improved stability or delivery of the molecule. Modified segmented miRNA mimetics can also minimize the possibility of activating an interferon response in, for example, humans.

In certain embodiments, segmented miRNA mimetics of the invention comprise ribonucleotides at about 1 or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26) of the nucleotide positions in one strand, in each strand, or any combination thereof.

In related embodiments, a segmented miRNA mimetic according to the instant disclosure comprises one or more natural or synthetic non-standard nucleotides. In related embodiments, the non-standard nucleotide is one or more deoxyuridine, L- or D-locked nucleic acid (LNA) molecule (e.g., a 5-methyluridine LNA) or substituted LNA (e.g., having a pyrene), or a universal-binding nucleotide, or a G clamp, or any combination thereof. In certain embodiments, the universal-binding nucleotide can be C-phenyl, C-naphthyl, inosine, azole carboxamide, 1-β-D-ribofuranosyl-4-nitro indole, 1-β-D-ribofuranosyl-5-nitroindole, 1-β-D-ribofuranosyl-6-nitroindole, or 1-β-D-ribofuranosyl-3-nitropyrrole.

Modified nucleotides, which can be present in either or both the passenger and the guide strands of a segmented miRNA mimetic of the invention, comprise modified nucleotide analogs having characteristics similar to natural or standard ribonucleotides. For example, this disclosure features segmented miRNA mimetics comprising nucleotides having a Northern conformation (see, e.g., Northern pseudorotation cycle, Saenger, Springer-Verlag ed., 1984), which are known to potentially impart resistant to nuclease degradation while maintaining the capacity to mediate RNAi, at least when applied to siRNA molecules. Exemplary nucleotides having a Northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-methoxyethyl (MOE) nucleotides, 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, 5-methyluridines, or 2′-O-methyl nucleotides). In any of these embodiments, one or more substituted or modified nucleotides can be a G clamp (e.g., a cytosine analog that forms an additional hydrogen bond to guanine, such as 9-(aminoethoxy)phenoxazine). See, e.g., Lin and Mateucci, 1998 J. Am. Chem. Soc. 720:8531).

In certain embodiments, a segmented miRNA mimetic of the invention comprises an overhang of 1 to 5 nucleotides. The overhang can comprise one or more 2′-O-alkyl modifications or locked nucleic acid (LNAs) as described herein or otherwise known in the art. In certain embodiments, a segmented miRNA mimetic of the invention can comprise one or more 3′-end 2′-O-alkyl or LNA at one or more of the internal terminals. A 2′-O-alkyl or LNA can also be present at positions that are not in the gaps, near the nicks or at the terminal ends of a segmented miRNA mimetic. In any of the embodiments of segmented miRNA mimetics, the 3′-terminal overhangs, if present, can comprise chemically-modified nucleotides that are modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of segmented miRNA mimetics, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of segmented miRNA mimetics, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.

In certain embodiments, the 5′-terminal end of the passenger strand or guide strand of a segmented miRNA mimetic of the invention is phosphorylated. In any of the embodiments of segmented miRNA mimetics described herein, the segmented miRNA can further comprise a terminal phosphate group, such as a 5′-phosphate (see Martinez et al., 2002 Cell 110:563; Schwarz et al., 2002 Mole. Cell 70:537) or a 5′3′-diphosphate.

In certain embodiments, a segmented miRNA mimetic comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) 2′-sugar substitutions in one strand or independently each strand, such as a 2′-deoxy, 2′-O-2-methoxyethyl, 2′-O-methoxyethyl, 2′-O-methyl, 2′-halogen (e.g., 2′-fluoro), 2′-O-allyl, or the like, or any combination thereof. In still further embodiments, a segmented miRNA mimetic comprises a terminal cap substituent at one or more terminal ends, internal ends, or both, of the passenger strand and/or the guide strands, such as, for example, an alkyl, abasic, deoxy abasic, glyceryl, dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety, or any combination thereof. In certain embodiments, at least one or more 5′-terminal-end or 5′-internal-end ribonucleotides of the passenger strand have 2′-sugar substitutions. In certain other embodiments, at least one or more 5′-terminal-end or 5′-internal-end ribonucleotides of the guide strand have 2′-sugar substitutions. In certain embodiments, at least one or more 5′-terminal-end or 5′-internal-end ribonucleotides of the passenger strand and the guide strand have 2′-sugar substitutions.

In other embodiments, a segmented miRNA mimetic comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) substitutions in the sugar in one strand or independently each strand, including any combination of ribosyl, 2′-deoxyribosyl, a tetrofuranosyl (e.g., L-α-threofuranosyl), a hexopyranosyl (e.g., β-allopyranosyl, β-altropyranosyl and β-glucopyranosyl), a pentopyranosyl (e.g., β-ribopyranosyl, α-lyxopyranosyl, β-xylopyranosyl and α-arabinopyranosyl), a carbocyclic analog, a pyranose, a furanose, a morpholino, or analogs or derivatives thereof.

In yet other embodiments, a segmented miRNA mimetic comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) modified internucleoside linkages in one strand or independently each strand, such as independently a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate, 3′-alkylene phosphonate, 5′-alkylene phosphonate, chiral phosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate, phosphoramidate, 3′-amino phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, boranophosphate linkage, or any combination thereof.

A modified internucleotide linkage, as described herein, can be present in one or more strands of a segmented miRNA mimetic, for example, in the passenger strand, the guide strand, or in both strands. A segmented miRNA mimetic can comprise one or more modified internucleotide linkages at the 3′-terminal end, the 5′-terminal end, or both of the 3′-terminal and 5′-terminal ends of the passenger strand, the guide strand, or both strands. In certain embodiments, a segmented miRNA mimetic of the invention has one modified internucleotide linkage at the 3′-terminal end, such as a phosphorothioate linkage. An exemplary segmented miRNA mimetic comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages in either strand. Another exemplary segmented miRNA mimetic comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages in both strands. A further exemplary segmented miRNA mimetic comprises about 1 to about 5 or more consecutive phosphorothioate internucleotide linkages at for example, the 5′-terminal end of its passenger strand, the 5′-terminal end of its guide strand, both the 5′-terminal ends of both strands, or for example, at one or more of the 5′-internal ends. In yet another exemplary segmented miRNA mimetic, there can be one or more pyrimidine phosphorothioate internucleotide linkages in the passenger strand and/or the guide strand. In a further exemplary segmented miRNA mimetic, there can be one or more purine phosphorothioate internucleotide linkages in the passenger strand and/or the guide strand.

Many exemplary modified nucleotide bases or analogs thereof useful in segmented miRNA mimetics of the instant disclosure include 5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl, 2-propyl, or other alkyl derivatives of adenine and guanine; 8-substituted adenines and guanines (e.g., 8-aza, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, or the like); 7-methyl, 7-deaza, and 3-deaza adenines and guanines; 2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-methyl, 5-propynyl, 5-halo (e.g., 5-bromo or 5-fluoro), 5-trifluoromethyl, or other 5-substituted uracils and cytosines; and 6-azouracil. Further useful nucleotide bases can be found in Kurreck, 2003 Eur. J. Biochem. 270:1628; Herdewijn, 2000 Guide Nucleic Acid Develop. 10:297; Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990; U.S. Pat. No. 3,687,808, and similar references, all of which are incorporated by reference herein.

Certain modified nucleotide base moieties are also contemplated. These include 5-substituted pyrimidines; 6-azapyrimidines; and N-2, N-6, or O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine). Further, for example, 5-methyluridine and 5-methylcytosine substitutions are known to increase nucleic acid duplex stability, which can be combined with 2′-sugar modifications (e.g., 2′-O-methyl or 2′-methoxyethyl) or internucleoside linkages (e.g., phosphorothioate) that provide the desired nuclease resistance to the modified or substituted segmented miRNA mimetics.

In certain embodiments, at least one pyrimidine of a segmented miRNA mimetic of the invention is a locked nucleic acid (LNA) in the form of a bicyclic sugar. In a related embodiment, the LNA comprises a base substitution, such as a 5-methyluridine LNA or 2-thio-5-methyluridine LNA. In certain embodiments, a ribose of the pyrimidine nucleoside or the internucleoside linkage can be optionally modified.

In any of these embodiments, one or more modified nucleotides can be a G clamp (e.g., a cytosine analog that forms an additional hydrogen bond to guanine, such as 9-(aminoethoxy) phenoxazine). See, e.g., Lin and Mateucci, 1998 Nucleic Acids Res. 19:3111).

In addition, the terminal structure of segmented miRNA mimetics of this disclosure can comprise a stem-loop structure in which an end of one strand (e.g., the guide strand) of a segmented miRNA mimetic is connected by a linker nucleic acid, e.g., a linker RNA to an end of the opposite strand (e.g., the passenger strand). When linker segments are employed, there is no particular limitation in the length of the linker as long as it does not hinder pairing of the stem portion. For example, for stable pairing of the stem portion, the linker portion can have a clover-leaf tRNA structure. Even if the linker has a length that would hinder pairing of the stem portion, it is possible, for example, to construct the linker portion to include introns so that the introns are excised during processing of a precursor miRNA mimetic into a mature miRNA mimetic, thereby allowing pairing of the stem portion. In the case of a stem-loop dsRNA, either end (head or tail) of a segmented miRNA mimetic with no loop structure can comprise a low molecular weight RNA, for example, a natural RNA molecule such as a tRNA, rRNA or viral RNA, or an artificial RNA molecule.

A segmented miRNA mimetic of the invention can be constructed such that it takes on an overall circular structure, wherein the entire molecule is about 10 to about 60 nucleotides in length having from about 5 to about 26 base pairs (e.g., about 19 to about 21) wherein the circular oligonucleotide forms a dumbbell shaped structure having about 10 to about 26 base pairs and two loops. In certain embodiments, a circular segmented miRNA mimetic contains two loop motifs, wherein one or both loop portions are biodegradable.

In another aspect of this disclosure, the segmented miRNA mimetic structures of the invention and their potential of allowing more suitable types of chemical modification and allowing modification to a higher extent can be used to reduce interferon activation when a segmented miRNA mimetic is contacted with a biological sample, for example, when it is introduced into a eukaryotic cell. A segmented miRNA mimetic of the invention comprises at least 6 ends, including terminal and internal ends, as compared to its traditional non-segmented duplex miRNA mimetic counterpart, which comprises 4 ends. These ends can conveniently be used for tethering functional chemical groups to enhance, for example, lipophilic and other properties associated with cellular delivery. For instance, it is possible to tether bulky groups like cholesterol to the 5′-ends of each of the contiguous stretches of nucleotides without losing RNAi activity.

Moreover, because the yield of synthesis is usually higher for shorter RNA strands, the cost of large-scale synthesis in connection with therapeutic application can be substantially reduced using the segmented miRNA mimetics of the present invention.

In any of the embodiments described herein, a segmented miRNA mimetic can include multiple types of modifications in combination. For example, a segmented miRNA mimetic having at least one ribothymidine or 2-thioribothymidine can further comprise at least one LNA, 2′-methoxy, 2′-fluoro, 2′-deoxy, phosphorothioate linkage, an inverted base terminal cap, or any combination thereof. In certain exemplary embodiments, a segmented miRNA mimetic can comprise one or more or all uridines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substituted with 2′-O-methyl uridine and have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LNA substitutions. In other exemplary embodiments, a segmented miRNA mimetic can comprise from one or more or all uridines substituted with 2′-O-methyl uridine and have up to about 25% phosphorothioate substitutions. In still other exemplary embodiments, a segmented miRNA mimetic can comprise one or more or all uridines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substituted with 2′-O-methyl uridine and have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2′-deoxy-2′-fluoro substitutions.

Within certain aspects, the present disclosure also provides segmented miRNA molecules comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base nucleotides. In certain aspects, a segmented miRNA mimetic disclosed herein can include about 1 to about 10 universal base nucleotides, so long as the resulting segmented miRNA mimetic remains capable of modulating one or more of its endogenous miRNA targets.

Suitable modifications can also include one or more suitable conjugates attached to, typically the ends, including the terminal ends and internal ends, of a segmented miRNA mimetic of the invention. The conjugate can be attached via a covalent attachment. In some cases, the conjugate can be linked to the segmented miRNA mimetic via a biodegradable linker, attached the 3′-end, 5′ end, or both ends of the passenger strand, the guide strand and/or the internal ends of each of the contiguous stretches of nucleotides. The conjugate molecule can facilitate the delivery of the double-stranded oligonucleotide molecule into a biological system, such as a cell. The conjugate can also be a polyethylene glycol (PEG), human serum albumin, or a ligand for a cellular receptor that can facilitate cellular uptake. However, as explained above, the endogenous miRNA and siRNA paths of biogenesis and machineries are distinct, featuring different components or participants, therefore conjugates or other modifications in this class that are suitable for an exogenously introduced siRNA molecule can still be unsuitable for an exogenously introduced miRNA mimetic molecule such as a segmented miRNA mimetic of the invention.

Substitutions and Insertions

Various non-nucleotide moieties as are provided herein or otherwise known in the art can be used as substitutions and/or insertions in the segmented miRNA mimetics of the invention provided that RNAi activity against one or more miRNA targets is maintained.

In one aspect of the invention, substitutions and/or insertions in the segmented mimetic miRNAs of the invention can comprise one or more alkyl moieties, e.g., any C₁-C₂₀, and preferably a C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉ or C₁₀ alykl moiety. The alykl moiety can be straight chain, branched, aliphatic or aromatic. The alykl moiety can be substituted or unsubstituted. In certain embodiments the alkyl moieties are C₃ and/or C₆.

Segmented mimetic miRNAs of the present invention can comprise substitutions or insertions that incorporate one or more small molecules, lipids or lipophiles, terpenes, phospholipids, antibodies, toxins, cholesterol, a protein binding agent (e.g., a ligand for a cellular receptor that can facilitate cellular uptake), a vitamin, negatively charged polymers and other polymers, for example proteins (e.g., human serum albumin), peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, and those described in, for example, U.S. Patent Publication No. 2005/0196781, and U.S. Patent Publication No. 2006/0293271, the disclosures of which are incorporated herein by reference. Substitutions and insertions can include alkyl chains optionally substituted with a functional group. For example, the alkyl chain can be substituted with a moiety that binds specifically to a target molecule of interest.

Substitutions and insertions of the present invention can further comprise a polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, 1990, Nucleic Acids Res. 18:6353; Seela and Kaiser, 1987, Nucleic Acids Res. 15:3113; Cload and Schepartz, 1991, J. Am. Chem. Soc. 113:6324; Richardson and Schepartz, 1991, J. Am. Chem. Soc. 113:5109; Ma et al., 1993, Nucleic Acids Res. 27:2585; Ma et al., 1993, Biochemistry 32:1751; Durand et al., 1990, Nucleic Acids Res. 18:6353; McCurdy et al., 1991, Nucleosides & Nucleotides 70:287; Jaschke et al., 1993, Tetrahedron Lett. 34:301; Ono et al., 1991, Biochemistry 30:9914; and others. A chemical moiety that provides additional functionality (e.g., specifically binds to a target molecule of interest or facilitates/enhances cellular delivery of the molecule) to the miRNA mimetic can be a part of the substitution or insertion or covalently attached or linked thereto. For example, the additional functional group can impart therapeutic activity to miRNA mimetic of the invention by assisting in transferring the RNAi molecule compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of RNAi molecules of the invention.

Substitutions and insertions of the present invention can aid in delivery and/or localization of RNAi molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). For example, the conjugate member can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, or a C5 pyrimidine linker. The conjugate member can be a glyceride lipid conjugate (e.g., a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols. The conjugate molecule can alternatively be a peptide that functions, when conjugated to a miRNA mimetic, to facilitate delivery of the molecule into a target cell, or otherwise enhance delivery, stability, or activity of the molecule when contacted with a biological sample. Exemplary peptide conjugate members for use within these aspects of this disclosure, include peptides PN27, PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404, PN453, and PN509 as described, for example, in U.S. Patent Application Publication Nos. 2006/0040882 and 2006/0014289, and U.S. Provisional Patent Application No. 60/939,578, which are all incorporated herein by reference.

A substitution or insertion can comprise a moiety that specifically binds to a target molecule. The target molecule can be any molecule of interest. For example, the target molecule can be a ligand-binding domain of a protein, thereby preventing or competing with the interaction of the naturally-occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, e.g., Gold et al, 1995, Annu. Rev. Biochem. 64:163; Brody and Gold, 2000, J. Biotechnol. 74:5; Sun, 2000, Curr. Opin. MoI. Ther. 2:100; Kusser, J., 2000, Biotechnol. 74:21; Hermann and Patel, 2000, Science 257:820; and Jayasena, 1999, Clinical Chem. 45:1628).

The substitution or insertion can provide the ability to administer the segmented miRNA mimetic to specific cell types, such as hepatocytes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262:4429) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell 22: 611; Connolly et al., 1982, J. Biol. Chem. 257:939). Lee and Lee (1987, Glycoconjugate J. 4:317) obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem. 24: 1388). The use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of bioconjugates of this disclosure.

Terminal Analogs, Modifications, Linkers, and Conjugates

In one embodiment, a miRNA mimetic of the invention comprises one or more terminal nucleotide analogs, non-nucleotide analogs, nucleotide linkers, non-nucleotide linkers, caps, conjugates and the like as are generally known in the art, at the 5′-end, 3′-end, or both 5′- and 3′-ends of the passenger strand, or alternately at the 3′-end of the guide strand.

In certain embodiments, the invention features a nucleic acid linker that covalently attaches on strand to the other. A nucleotide linker can be a nucleic acid aptamer. A non-nucleotide linker can be an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, 1990 Nucleic Acids Res. 18:6353; Seela and Kaiser, 1987 Nucleic Acids Res. 15:3113; Cload and Schepartz, 1991 J. Am. Chem. Soc. 113:6324; Richardson and Schepartz, 1991 J. Am. Chem. Soc. 113:5109; Ma et al., 1993 Nucleic Acids Res. 27:2585; Ma et al., 1993 Biochemistry 32:1751; Durand et al., 1990 Nucleic Acids Res. 18:6353; McCurdy et al., 1991 Nucleosides & Nucleotides 70:287; Jaschke et al., 1993 Tetrahedron Lett. 34:301; Ono et al., 1991 Biochemistry 30:9914; and others.

In another embodiment, a conjugate molecule can be optionally attached to a segmented miRNA mimetic or an analog thereof. For example, such conjugate molecules can be polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can, for example, mediate cellular uptake. The conjugate molecule can be attached at one or more of the terminal ends and/or one or more of the internal ends. Examples of specific conjugate molecules contemplated by the instant disclosure are described in, for example, U.S. Patent Publication No. 2005/0196781 A1, and U.S. Patent Publication No. 2006/0293271 A1, the disclosures of which are incorporated herein by reference.

In a certain aspect, the invention features conjugates and/or complexes of segmented miRNA mimetics of the invention. Such conjugates and/or complexes can be used to facilitate delivery of one or more segmented miRNA mimetics to a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of segmented miRNA mimetics of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates described herein can be attached to biologically active segmented miRNA mimetics via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

A person of skill in the art can screen segmented miRNA mimetics of this disclosure having various conjugates to determine which of the segmented miRNA-conjugate complexes possess improved properties (e.g., pharmacokinetic profile, bioavailability, stability) while maintaining the ability to mediate RNAi in, for example, an animal model as described herein or generally known in the art.

In another aspect, a segmented miRNA mimetic of the invention comprises one or more 5′- and/or a 3′-cap structures, for example at the terminal ends of the passenger strand, guide strand, both strands, or any of the internal ends of the contiguous stretches of nucleotides. In non-limiting examples: a suitable 5′-cap can be one selected from the group comprising inverted abasic residue (moiety); LNA; 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.

In another non-limiting example, a suitable 3′-cap can be selected from a group comprising, LNA; 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties. For more details, see Beaucage and Iyer, 1993, Tetrahedron 49:1925, which is incorporated by reference herein.

Making microRNA Mimetics of the Invention

Exemplary molecules of the instant disclosure can be recombinantly produced (e.g., isolated), chemically synthesized, or a combination thereof. Oligonucleotides or individual contiguous stretches of nucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example, as described in Caruthers et al., 1992 Methods in Enzymol. 211:3; Thompson et al, PCT Publication No. WO 99/54459, Wincott et al., 1995 Nucleic Acids Res. 23:2677; Wincott et al., 1997 Methods MoI. Bio. 74:59; Brennan et al., 1998 Biotechnoh Bioeng. 67:33; and Brennan, U.S. Pat. No. 6,001,311. Synthesis of RNA, including certain segmented miRNA mimetics thereof of this disclosure, can be made using the procedure as described in Usman et al., 1987 J. Am. Chem. Soc. 109:7845; Scaringe et al., 1990 Nucleic Acids Res. 18:5433; and Wincott et al., 1995 Nucleic Acids Res. 23:2677; Wincott et al., 1997 Methods MoI. Bio. 74:59. In certain embodiments, segmented miRNA mimetics of the present disclosure can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992 Science 256:9923; Draper et al., PCT Publication No. WO 93/23569; Shabarova et al., 1991 Nucleic Acids Res. 19:4247; Bellon et al., 1997 Nucleosides & Nucleotides 16:951; Bellon et al., 1997 Bioconjugate Chem. 8:204), or by hybridization following synthesis or deprotection. In certain embodiments, a segmented miRNA mimetic of this disclosure can be made as single or multiple transcription products expressed by a polynucleotide vector encoding one or more contiguous stretches of RNAs and directing their expression within host cells. In all of the embodiments herein, the double-stranded portion of a final transcription product to be expressed within the target cell can be, for example, about 10 to about 26 bp, about 12 to about 25 bp, or about 14 to about 22 bp long.

Methods for Designing a Segmented miRNA Mimetic

As described herein, a segmented miRNA mimetic can be designed based on a corresponding non-segmented duplex miRNA mimetic molecule, which is in turn designed based on known endogenous miRNA molecules, such as those listed in the miRBase as of the filing date of the present application, and for example, SEQ ID Nos: 1-1090 in Table I. The segmented miRNA mimetic is then characterized as described below and in the Examples herein.

Specifically, any segmented miRNA mimetic of the invention can be designed by introducing one or more discontinuities of the invention (nicks, gaps, substitutions, and/or insertions) into the passenger strand, the guide strand, or both the passenger and the guide strands. The discontinuity can be introduced at the 5′-end of any of position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, and/or position 26 of the guide strand. The discontinuity can be introduced at the 5′-end of any of position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, and/or position 26 of the passenger strand.

In certain embodiments, a method is provided wherein one or more genes, which are known to be regulated by an endogenous miRNA, are selected to indicate the RNAi potency of a segmented miRNA mimetic. The RNAi activity of a given segmented miRNA mimetic can be measured using known methods, such as those described generally in Fire et al., PCT Publication No. WO99/32619. In some embodiments, the instant specification provides methods for selecting more efficacious segmented miRNA mimetic designs by using one or more reporter gene constructs comprising a constitutive promoter, such as a cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter, operably fused to, and capable of altering the expression of one or more reporter genes, such as a luciferase, chloramphenicol (CAT), or β-galactosidase, which, in turn, is operably fused in-frame with a segmented miRNA mimetic. These reporter gene expression constructs can be co-transfected with one or more segmented miRNA mimetics and the control (corresponding) non-segmented miRNA mimetics. The capacity of a given segmented miRNA mimetic to mediate RNAi of a target mRNA can be determined by comparing the measured reporter gene activity in cells transfected with the segmented miRNA mimetic and the activity in cells transfected with a negative control (i.e., in cells not transfected with the segmented miRNA mimetic) and a positive control (i.e., in cells transfected with the corresponding non-segmented duplex miRNA mimetic). The segmented miRNA mimetics having at least 20% or more, preferably at least 40% or more, or 60% or more, or 80% or more, of the activity of their corresponding non-segmented duplex miRNA mimetics are selected.

Certain embodiments disclosed herein also provide methods for selecting one or more segmented miRNA mimetics based on their predicted stability. A theoretical melting curve can be prepared for each of the segmented miRNA mimetic designs such that those with high theoretical melting curves, and therefore higher duplex stability and corresponding lower cytotoxic effects, would be selected. Alternatively, stability of a segmented miRNA mimetic can be determined empirically and those with higher measured melting temperatures would be selected.

Compositions and Methods of Use

As set forth herein, segmented miRNA mimetics of the invention are miRNA mimetics that are designed to supplement or take the place of corresponding naturally-occurring miRNAs, the reduced or otherwise unsuitably low levels of which have been associated with pathological or diseased conditions. A segmented miRNA mimetic of the invention is therefore preferably capable of participating in the cellular RNAi pathway or otherwise capable of modulating the same or related pathway(s). A segmented miRNA mimetic of the invention can be introduced to a cell, tissue, organism, an in vitro, or an in vivo system to mediate RNAi against an endogenous RNA target of its corresponding naturally-occurring miRNA. As such, a segmented miRNA mimetic can regulate a number of genes, for example, downstream from its RNA target, whose expression levels are associated with or otherwise regulated by the corresponding naturally-occurring miRNA. Because aberrant expression levels of certain naturally-occurring miRNAs have been implicated in various human ailments, including, but are not limited to, hyperproliferative, angiogenic, or inflammatory diseases, states, or adverse conditions, the segmented miRNA mimetics of the present invention can offer valuable therapeutic opportunities. In this context, a segmented miRNA mimetic of this disclosure can regulate (e.g., knockdown or up-regulate) expression of one or more downstream genes of its corresponding endogenous miRNA, such that prevention, alleviation, or reduction of the severity or recurrence of one or more associated disease symptoms can be achieved. Alternatively, for various distinct disease models in which expression of one or more target mRNAs are not necessarily reduced or at a lower-than-normal level as a consequence or sequel of diseases or other adverse conditions, introducing exogenous miRNA mimetics, such as one or more segmented miRNA mimetics of the invention, can nonetheless result in a therapeutic result by affecting the expression levels of genes associated with the disease pathway. The segmented miRNA mimetics of the invention thus are useful reagents, which can be in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

In certain embodiments, aqueous suspensions containing one or more segmented miRNA mimetics of the invention can be prepared in admixture with suitable excipients, such as suspending agents or dispersing or wetting agents. Exemplary suspending agents include sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia. Representative dispersing or wetting agents include naturally-occurring phosphatides (e.g., lecithin), condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). In certain embodiments, the aqueous suspensions can optionally contain one or more preservatives (e.g., ethyl or w-propyl-p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, or one or more sweetening agents (e.g., sucrose, saccharin). In additional embodiments, dispersible powders and granules suitable for preparation of an aqueous suspension comprising one or more segmented miRNA mimetics of the invention can be prepared by the addition of water with the segmented miRNA mimetics in admixture with a dispersing or wetting agent, suspending agent and optionally one or more preservative, coloring agent, flavoring agent, or sweetening agent. The present disclosure also includes segmented miRNA mimetic compositions prepared for storage or administration that include a pharmaceutically effective amount of a desired compound in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., A. R. Gennaro edit., 21st Edition, 2005. In certain embodiments, pharmaceutical compositions of this disclosure can optionally include preservatives, antioxidants, stabilizers, dyes, flavoring agents, or any combination thereof. Exemplary preservatives include sodium benzoate, esters of p-hydroxybenzoic acid, and sorbic acid.

The segmented miRNA mimetic compositions of the instant disclosure can be effectively employed as pharmaceutically-acceptable formulations. Pharmaceutically-acceptable formulations prevent, alter the occurrence or severity of, or treat (alleviate one or more symptom(s) to a detectable or measurable extent) a disease state or other adverse condition in a subject. A pharmaceutically acceptable formulation includes salts of the above compounds, for example, acid addition salts, such as salts of hydrochloric acid, hydrobromic acid, acetic acid, or benzene sulfonic acid. A pharmaceutical composition or formulation refers to a composition or formulation in a form suitable for administration into a cell, or a subject such as a human (e.g., systemic administration). The formulations of the present disclosure, having an amount of segmented miRNA mimetic sufficient to treat or prevent a disorder associated with target gene expression are, for example, suitable for topical (e.g., creams, ointments, skin patches, eye drops, ear drops) application or administration. Other routes of administration include oral, parenteral, sublingual, bladder washout, vaginal, rectal, enteric, suppository, nasal, and inhalation. The pharmaceutical compositions of the present disclosure are formulated to allow the segmented miRNA mimetic contained therein to be bioavailable upon administration to a subject.

In certain embodiments, a segmented miRNA of this disclosure can be formulated as oily suspensions or emulsions (e.g., oil-in-water) by suspending the segmented miRNA mimetic in, for example, a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil) or a mineral oil (e.g., liquid paraffin). Suitable emulsifying agents can be naturally-occurring gums (e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides (e.g., soy bean, lecithin, esters or partial esters derived from fatty acids and hexitol), anhydrides (e.g., sorbitan monooleate), or condensation products of partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). In certain embodiments, the oily suspensions or emulsions can optionally contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. In related embodiments, sweetening agents and flavoring agents can optionally be added to provide palatable oral preparations. In yet other embodiments, these compositions can be preserved by the optionally adding an anti-oxidant, such as ascorbic acid.

In certain embodiments, a segmented miRNA mimetic can be formulated as syrups and elixirs with sweetening agents (e.g., glycerol, propylene glycol, sorbitol, glucose or sucrose). Such formulations can also contain a demulcent, preservative, flavoring, coloring agent, or any combination thereof. In other embodiments, pharmaceutical compositions comprising a segmented miRNA mimetic can be in the form of a sterile, injectable aqueous or oleaginous suspension. The sterile injectable preparation can also be a sterile, injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., as a solution in 1,3-butanediol). Among the exemplary acceptable vehicles and solvents useful in the compositions of this disclosure is water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile, fixed oils can be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of parenteral formulations.

Within certain embodiments of this disclosure, pharmaceutical compositions and methods are provided that feature the presence or administration of one or more segmented miRNA mimetics, combined, complexed, or conjugated with a polypeptide, optionally formulated with a pharmaceutically-acceptable carrier, such as a diluent, stabilizer, buffer, or the like. The negatively charged segmented miRNA mimetics can be administered to a patient by any standard means, with or without stabilizers, buffers, or the like, to form a composition suitable for treatment. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present disclosure can also be formulated and used as a tablet, capsule or elixir for oral administration, suppository for rectal administration, sterile solution, or suspension for injectable administration, either with or without other compounds known in the art. Thus, a segmented miRNA mimetic of the present disclosure can be administered in any form, such as nasally, transdermally, parenterally, or by local injection.

In accordance with this disclosure herein, a segmented miRNA mimetic (optionally substituted or modified or conjugated), compositions thereof, and methods for inhibiting expression of one or more corresponding target mRNAs in a cell or organism are provided. In certain embodiments, this disclosure provides methods and segmented miRNA mimetic compositions for treating a subject, including a human cell, tissue or individual, having a disease or at risk of developing a disease caused by or associated with the aberrant levels of its corresponding naturally-occurring miRNA. In a certain embodiment, the method includes administering a segmented miRNA mimetic or a pharmaceutical composition containing the segmented miRNA mimetic to a cell or an organism, such as a mammal, such that the level of its corresponding naturally-occurring miRNA within the cell or the organism is increased. Subjects (e.g., mammalian, human) amendable for treatment using the segmented miRNA mimetics (optionally substituted or modified or conjugated), compositions thereof, and methods of the present disclosure include those suffering from one or more disease or condition mediated, at least in part, by an aberrant expression level of its corresponding naturally-occurring miRNA, or which are amenable to treatment by replenishing or increasing the level of RNAi mediated by the corresponding miRNA, including a hyperproliferative (e.g., cancer), angiogenic, metabolic, or inflammatory (e.g., arthritis) disease or disorder or condition.

Compositions and methods disclosed herein are useful in the treatment of a wide variety of target viruses, including retrovirus, such as human immunodeficiency virus (HIV), Hepatitis C Virus, Hepatitis B Virus, Coronavirus, as well as respiratory viruses, including human Respiratory Syncytial Virus, human Metapneumovirus, human Parainfluenza virus Rhinovirus and Influenza virus.

In other examples, the compositions and methods of this disclosure are useful as therapeutic tools to treat or prevent symptoms of, for example, hyperproliferative disorders. Exemplary hyperproliferative disorders include neoplasms, carcinomas, sarcomas, tumors, or cancer. More exemplary hyperproliferative disorders include oral cancer, throat cancer, laryngeal cancer, esophageal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, gastrointestinal tract cancer, gastrointestinal stromal tumors (GIST), small intestine cancer, colon cancer, rectal cancer, colorectal cancer, anal cancer, pancreatic cancer, breast cancer, cervical cancer, uterine cancer, vulvar cancer, vaginal cancer, urinary tract cancer, bladder cancer, kidney cancer, adrenocortical cancer, islet cell carcinoma, gallbladder cancer, stomach cancer, prostate cancer, ovarian cancer, endometrial cancer, trophoblastic tumor, testicular cancer, penial cancer, bone cancer, osteosarcoma, liver cancer, extrahepatic bile duct cancer, skin cancer, basal cell carcinoma (BCC), lung cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), brain cancer, melanoma, Kaposi's sarcoma, eye cancer, head and neck cancer, squamous cell carcinoma of head and neck, tymoma, thymic carcinoma, thyroid cancer, parathyroid cancer, Hippel-Lindau syndrome, leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, hairy cell leukemia, lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, T-cell lymphoma, multiple myeloma, malignant pleural mesothelioma, Barrett's adenocarcinoma, Wilm's tumor, or the like. In other examples, the compositions and methods of this disclosure are useful as therapeutic tools to regulate expression of one or more target gene to treat or prevent symptoms of, for example, inflammatory disorders. Exemplary inflammatory disorders include diabetes mellitus, rheumatoid arthritis, pannus growth in inflamed synovial lining, collagen-induced arthritis, spondylarthritis, ankylosing spondylitis, multiple sclerosis, encephalomyelitis, inflammatory bowel disease, Chron's disease, psoriasis or psoriatic arthritis, myasthenia gravis, systemic lupus erythematosis, graft-versus-host disease, atherosclerosis, and allergies.

Other exemplary disorders that can be treated with segmented miRNA mimetics, compositions and methods of the instant disclosure include metabolic disorders, cardiac disease, pulmonary disease, neovascularization, ischemic disorders, age-related macular degeneration, diabetic retinopathy, glomerulonephritis, diabetes, asthma, chronic obstructive pulmonary disease, chronic bronchitis, lymphangiogenesis, and atherosclerosis.

Within additional aspects, combination formulations and methods are provided comprising an effective amount of one or more segmented miRNA mimetics in combination with one or more secondary or adjunctive active agents that are formulated together or administered coordinately with the segmented miRNA mimetics of the invention to control one or more target gene-associated disease or condition as described herein. Useful adjunctive therapeutic agents in these combinatorial formulations and coordinate treatment methods include, for example, enzymatic nucleic acid molecules, allosteric nucleic acid molecules, guide, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules and other organic or inorganic compounds including metals, salts and ions, and other drugs and active agents indicated for treating one or more target gene-associated disease or condition, including chemotherapeutic agents used to treat cancer, steroids, non-steroidal anti-inflammatory drugs (NSAIDs), or the like. Exemplary chemotherapeutic agents include alkylating agents (e.g., cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogen mustards, uramustine, temozolomide), antimetabolites (e.g., aminopterin, methotrexate, mercaptopurine, fluorouracil, cytarabine), taxanes (e.g., paclitaxel, docetaxel), anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idaruicin, mitoxantrone, valrubicin), bleomycin, mytomycin, actinomycin, hydroxyurea, topoisomerase inhibitors (e.g., camptothecin, topotecan, irinotecan, etoposide, teniposide), monoclonal antibodies (e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab, rituximab, tositumomab, trastuzumab), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), cyclophosphamide, prednisone, leucovorin, oxaliplatin. To practice the coordinate administration methods of this disclosure, a segmented miRNA mimetic is administered simultaneously or sequentially in a coordinated treatment protocol with one or more secondary or adjunctive therapeutic agents described herein or known in the art. The coordinate administration can be done in either order, and there can be a time period while only one or both (or all) active therapeutic agents, individually or collectively, exert their biological activities. A distinguishing aspect of all such coordinate treatment methods is that the segmented miRNA mimetic present in a composition elicits some favorable clinical response, which can or can not be in conjunction with a secondary clinical response provided by the secondary therapeutic agent. For example, the coordinate administration of a segmented miRNA mimetic with a secondary therapeutic agent as contemplated herein can yield an enhanced (e.g., synergistic) therapeutic response beyond the therapeutic response elicited by either or both the purified segmented miRNA mimetic and the secondary therapeutic agent alone.

In another embodiment, a segmented miRNA mimetic of this disclosure can include a conjugate member on one or more of the nucleotides, at the terminal positions or the internal positions. The conjugate member can be, for example, a lipophile, a terpene, a protein binding agent, a vitamin, a carbohydrate, or a peptide. For example, the conjugate member can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, or a C5 pyrimidine linker. In other embodiments, the conjugate member is a glyceride lipid conjugate (e.g., a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols. Additional conjugate members include peptides that function, when conjugated to a modified segmented miRNA mimetic, to facilitate delivery of the mimetic into a target cell, or otherwise enhance delivery, stability, or activity of the mimetic when contacted with a biological sample. Exemplary peptide conjugate members for use within these aspects of this disclosure, include peptides PN27, PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404, PN453, and PN509 are described, for example, in U.S. Patent Application Publication Nos. 2006/0040882 and 2006/0014289, and U.S. Provisional Patent Application No. 60/939,578, which are all incorporated herein by reference. In certain embodiments, when peptide conjugate partners are used to enhance delivery of one or more segmented miRNA mimetics of this disclosure, the resulting formulations and methods will often exhibit further reduction of an interferon response in target cells as compared to a segmented miRNA mimetic delivered in combination with alternate delivery vehicles, such as lipid delivery vehicles (e.g., Lipofectamine™). In still another embodiment, a segmented miRNA mimetic of the invention can be conjugated to a polypeptide and admixed with one or more non-cationic lipids or a combination of a non-cationic lipid and a cationic lipid to form a composition that enhances intracellular delivery of the segmented miRNA mimetic as compared to delivery resulting from contacting the target cells with a naked segmented miRNA mimetic without the lipids. In more detailed aspects of this disclosure, the mixture, complex or conjugate comprising a segmented miRNA mimetic and a polypeptide can be optionally combined with (e.g., admixed or complexed with) a cationic lipid, such as Lipofectine™. To produce these compositions comprised of a polypeptide, a segmented miRNA mimetic and a cationic lipid, the segmented miRNA mimetic and the polypeptide can be mixed together first in a suitable medium such as a cell culture medium, after which the cationic lipid is added to the mixture to form an segmented miRNA mimetic/delivery peptide/cationic lipid composition. Optionally, the peptide and cationic lipid can be mixed together first in a suitable medium such as a cell culture medium, followed by the addition of the segmented miRNA mimetic to form the segmented miRNA mimetic/delivery peptide/cationic lipid composition.

This disclosure also features the use of segmented miRNA mimetic compositions comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations can offer increased accumulation of drugs in target tissues (Lasic et al., 1995 Chem. Rev., 95:2601; Ishiwata et al., 1995 Chem. Pharm. Bull. 43:1005). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., 1995 Science 267:1215; Oku et al., 1995 Biochim. Biophys. Acta 1238:86). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of nucleic acid molecules as compared to conventional cationic liposomes, which are known to accumulate in tissues of the mononuclear phagocytic system (MPS) (Liu et al., 1995 J. Biol. Chem. 42:24864; Choi et al., PCT Publication No. WO 96/10391; Ansell et al., PCT Publication No. WO 96/10390; Holland et al., PCT Publication No. WO 96/10392). Long-circulating liposomes can also provide additional protection from nuclease degradation as compared to cationic liposomes in theory due to avoiding accumulation in metabolically aggressive MPS tissues, such as the liver and spleen. In a certain embodiment, this disclosure provides compositions suitable for administering segmented miRNA mimetics of this disclosure to specific cell types, such as hepatocytes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987 J. Biol. Chem. 262:4429) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980 Cell 22: 611; Connolly et al., 1982 J. Biol. Chem. 257:939). Lee and Lee (1987 Glycoconjugate J. 4:317) obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981 J. Med. Chem. 24: 1388). The use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of bioconjugates of this disclosure.

The present disclosure also features a method for preparing segmented miRNA mimetic nanoparticles. A first solution containing melamine derivatives is dissolved in an organic solvent such as dimethyl sulfoxide, or dimethyl formamide to which an acid such as HCl has been added. The concentration of HCl would be about 3.3 moles of HCl for every mole of the melamine derivative. The first solution is then mixed with a second solution, which includes a nucleic acid dissolved or suspended in a polar or hydrophilic solvent (e.g., an aqueous buffer solution containing, for instance, ethylenediaminetraacetic acid (EDTA), or tris(hydroxymethyl)aminomethane (TRIS), or combinations thereof. The mixture forms a first emulsion. The mixing can be done using any standard technique such as, for example, sonication, vortexing, or in a micro fluidizer. The resultant nucleic acid particles can be purified and the organic solvent removed using size-exclusion chromatography or dialysis or both. The complexed nucleic acid nanoparticles can then be mixed with an aqueous solution containing either polyarginine or a Gln-Asn polymer, or both, in an aqueous solution. A preferred molecular weight of each polymer is about 5000 to about 15,000 Daltons. This forms a solution containing nanoparticles of nucleic acid complexed with the melamine derivative and the polyarginine and the Gln-Asn polymers. The mixing steps are carried out in a manner that minimizes shearing of the nucleic acid while producing nanoparticles on average smaller than about 200 nanometers in diameter. It is believed that the polyarginine complexes with the negative charge of the phosphate groups within the minor groove of the nucleic acid, and the polyarginine wraps around the trimeric nucleic acid complex. At either terminus of the polyarginine other moieties, such as the TAT polypeptide, mannose or galactose, can be covalently bound to the polymer to direct binding of the nucleic acid complex to specific tissues, such as to the liver when galactose is used. While not being bound to theory, it is believed that the Gln-Asn polymer complexes with the nucleic acid complex within the major groove of the nucleic acid through hydrogen bonding with the bases of the nucleic acid. The polyarginine and the Gln-Asn polymer should be present at a concentration of 2 moles per every mole of nucleic acid having 20 base pairs. The concentration should be increased proportionally for a nucleic acid having more than 20 base pairs. For example, if the nucleic acid has 25 base pairs, the concentration of the polymers should be 2.5-3 moles per mole of double-stranded nucleic acid. The resultant nanoparticles can be purified by standard means such as size exclusion chromatography followed by dialysis. The purified complexed nanoparticles can then be lyophilized using techniques well known in the art. In certain embodiments of the present disclosure provides nanoparticles less than 100 nanometers (nm) comprising a segmented miRNA mimetic.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the specific subject under consideration for treatment, concurrent medication, and other factors that those skilled in the medical arts will recognize. For example, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients can be administered depending on the potency of a segmented miRNA mimetic of this disclosure.

Dosage levels in the order of about 0.1 mg to about 140 mg per kilogram of body weight per day can be useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. Following administration of a segmented miRNA mimetic composition according to the formulations and methods of this disclosure, test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated, as compared to placebo-treated or other suitable control subjects.

Nucleic acid molecules and polypeptides can be administered to cells or organisms by a variety of methods known to those of skill in the art, including administration of formulations that comprise a miRNA mimetic and/or a polypeptide alone, or formulations that further comprise one or more additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, or the like. In certain embodiments, a segmented miRNA mimetic of the invention, and/or the polypeptide can be encapsulated in liposomes, administered by iontophoresis, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (see, e.g., PCT Publication No. WO 00/53722). Alternatively, a nucleic acid/peptide/vehicle combination can be locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of this disclosure, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies, such as those described in Conroy et al, 1999 Clin. Cancer Res. 5:2330; and PCT Publication No. WO 99/31262.

A segmented miRNA mimetic of the invention can also be administered in the form of suppositories, for example, for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

Further methods for delivery of nucleic acid molecules, such as a segmented miRNA mimetic of this invention, have been described in, for example, Boado et al., 1998 J. Pharm. ScL, 87:1308; Tyler et al., 1999 FEBS Lett. 421:2m; Pardridge et al., 1995 Proc. Nat'l Acad. ScL USA 92:5592; Boado, 1995 Adv. Drug Delivery Rev. 15:73; Aldrian-Herrada et al. 1998 Nucleic Acids Res. 26:4910; Tyler et al., 1999 Proc. Nat'l Acad. Sci. USA 96:7053; Akhtar et al., 1992 Trends Cell Bio. 2:139; “Delivery Strategies for Guide Oligonucleotide Therapeutics,” ed. Akhtar, 1995, Maurer et al., 1999 MoI. Membr. Biol. 16:129; Lee et al., 2000 ACS Symp. Ser., 752:184. In addition to in vivo and therapeutic applications, a skilled person in the art will appreciate that the segmented miRNA mimetics of the present disclosure are useful in a wide variety of in vitro applications, such as in scientific and commercial research (e.g., elucidation of physiological pathways, drug discovery and development), and medical and veterinary diagnostics.

All U.S. patents, U.S. patent publications, U.S. patent applications, foreign patents, foreign patent applications, non-patent publications, figures, and websites referred to in this specification are expressly incorporated herein by reference, in their entirety.

Table I lists certain endogenous mammalian and viral miRNA sequences, wherein the seed sequences, confirmed or projected, are capitalized. All miRNA sequences in Table I are derived from humans and are shown in 5′ to 3′ orientation. Other miRNA sequences of the present invention can be found in the miRBase, the content of which is incorporated by reference herein.

TABLE I  miRBase SEQ ID miRNA name number Sequence NO hsa-let-7a MIMAT0000062 UGAGGUAGuagguuguauaguu 1 hsa-let-7a* MIMAT0004481 CUAUACAAucuacugucuuuc 2 hsa-let-7a-2* MIMAT0010195 CUGUACAGccuccuagcuuucc 3 hsa-let-7b MIMAT0000063 UGAGGUAGuagguugugugguu 4 hsa-let-7b* MIMAT0004482 CUAUACAAccuacugccuuccc 5 hsa-let-7c MIMAT0000064 UGAGGUAGuagguuguaugguu 6 hsa-let-7c* MIMAT0004483 UAGAGUUAcacccugggaguua 7 hsa-let-7d MIMAT0000065 AGAGGUAGuagguugcauaguu 8 hsa-let-7d* MIMAT0004484 CUAUACGAccugcugccuuucu 9 hsa-let-7e MIMAT0000066 UGAGGUAGgagguuguauaguu 10 hsa-let-7e* MIMAT0004485 CUAUACGGccuccuagcuuucc 11 hsa-let-7f MIMAT0000067 UGAGGUAGuagauuguauaguu 12 hsa-let-7f-1* MIMAT0004486 CUAUACAAucuauugccuuccc 13 hsa-let-7f-2* MIMAT0004487 CUAUACAGucuacugucuuucc 14 hsa-miR-15a MIMAT0000068 UAGCAGCAcauaaugguuugug 15 hsa-miR-15a* MIMAT0004488 CAGGCCAUauugugcugccuca 16 hsa-miR-16 MIMAT0000069 UAGCAGCAcguaaauauuggcg 17 hsa-miR-16-1* MIMAT0004489 CCAGUAUUaacugugcugcuga 18 hsa-miR-17 MIMAT0000070 CAAAGUGCuuacagugcagguag 19 hsa-miR-17* MIMAT0000071 ACUGCAGUgaaggcacuuguag 20 hsa-miR-18a MIMAT0000072 UAAGGUGCaucuagugcagauag 21 hsa-miR-18a* MIMAT0002891 ACUGCCCUaagugcuccuucugg 22 hsa-miR-19a* MIMAT0004490 AGUUUUGCauaguugcacuaca 23 hsa-miR-19a MIMAT0000073 UGUGCAAAucuaugcaaaacuga 24 hsa-miR-19b-1* MIMAT0004491 AGUUUUGCagguuugcauccagc 25 hsa-miR-19b MIMAT0000074 UGUGCAAAuccaugcaaaacuga 26 hsa-miR-19b-2* MIMAT0004492 AGUUUUGCagguuugcauuuca 27 hsa-miR-20a MIMAT0000075 UAAAGUGCuuauagugcagguag 28 hsa-miR-20a* MIMAT0004493 ACUGCAUUaugagcacuuaaag 29 hsa-miR-21 MIMAT0000076 UAGCUUAUcagacugauguuga 30 hsa-miR-21* MIMAT0004494 CAACACCAgucgaugggcugu 31 hsa-miR-22* MIMAT0004495 AGUUCUUCaguggcaagcuuua 32 hsa-miR-22 MIMAT0000077 AAGCUGCCaguugaagaacugu 33 hsa-miR-23a* MIMAT0004496 GGGGUUCCuggggaugggauuu 34 hsa-miR-23a MIMAT0000078 AUCACAUUgccagggauuucc 35 hsa-miR-24-1* MIMAT0000079 UGCCUACUgagcugauaucagu 36 hsa-miR-24 MIMAT0000080 UGGCUCAGuucagcaggaacag 37 hsa-miR-24-2* MIMAT0004497 UGCCUACUgagcugaaacacag 38 hsa-miR-25* MIMAT0004498 AGGCGGAGacuugggcaauug 39 hsa-miR-25 MIMAT0000081 CAUUGCACuugucucggucuga 40 hsa-miR-26a MIMAT0000082 UUCAAGUAauccaggauaggcu 41 hsa-miR-26a-1* MIMAT0004499 CCUAUUCUugguuacuugcacg 42 hsa-miR-26b MIMAT0000083 UUCAAGUAauucaggauaggu 43 hsa-miR-26b* MIMAT0004500 CCUGUUCUccauuacuuggcuc 44 hsa-miR-27a* MIMAT0004501 AGGGCUUAgcugcuugugagca 45 hsa-miR-27a MIMAT0000084 UUCACAGUggcuaaguuccgc 46 hsa-miR-28-5p MIMAT0000085 AAGGAGCUcacagucuauugag 47 hsa-miR-28-3p MIMAT0004502 CACUAGAUugugagcuccugga 48 hsa-miR-29a* MIMAT0004503 ACUGAUUUcuuuugguguucag 49 hsa-miR-29a MIMAT0000086 UAGCACCAucugaaaucgguua 50 hsa-miR-30a MIMAT0000087 UGUAAACAuccucgacuggaag 51 hsa-miR-30a* MIMAT0000088 CUUUCAGUcggauguuugcagc 52 hsa-miR-31 MIMAT0000089 AGGCAAGAugcuggcauagcu 53 hsa-miR-31* MIMAT0004504 UGCUAUGCcaacauauugccau 54 hsa-miR-32 MIMAT0000090 UAUUGCACauuacuaaguugca 55 hsa-miR-32* MIMAT0004505 CAAUUUAGugugugugauauuu 56 hsa-miR-33a MIMAT0000091 GUGCAUUGuaguugcauugca 57 hsa-miR-33a* MIMAT0004506 CAAUGUUUccacagugcaucac 58 hsa-miR-92a-1* MIMAT0004507 AGGUUGGGaucgguugcaaugcu 59 hsa-miR-92a MIMAT0000092 UAUUGCACuugucccggccugu 60 hsa-miR-92a-2* MIMAT0004508 GGGUGGGGauuuguugcauuac 61 hsa-miR-93 MIMAT0000093 CAAAGUGCuguucgugcagguag 62 hsa-miR-93* MIMAT0004509 ACUGCUGAgcuagcacuucccg 63 hsa-miR-95 MIMAT0000094 UUCAACGGguauuuauugagca 64 hsa-miR-96 MIMAT0000095 UUUGGCACuagcacauuuuugcu 65 hsa-miR-96* MIMAT0004510 AAUCAUGUgcagugccaauaug 66 hsa-miR-98 MIMAT0000096 UGAGGUAGuaaguuguauuguu 67 hsa-miR-99a MIMAT0000097 AACCCGUAgauccgaucuugug 68 hsa-miR-99a* MIMAT0004511 CAAGCUCGcuucuaugggucug 69 hsa-miR-100 MIMAT0000098 AACCCGUAgauccgaacuugug 70 hsa-miR-100* MIMAT0004512 CAAGCUUGuaucuauagguaug 71 hsa-miR-101* MIMAT0004513 CAGUUAUCacagugcugaugcu 72 hsa-miR-101 MIMAT0000099 UACAGUACugugauaacugaa 73 hsa-miR-29b-1* MIMAT0004514 GCUGGUUUcauauggugguuuaga 74 hsa-miR-29b MIMAT0000100 UAGCACCAuuugaaaucaguguu 75 hsa-miR-29b-2* MIMAT0004515 CUGGUUUCacaugguggcuuag 76 hsa-miR-103-2* MIMAT0009196 AGCUUCUUuacagugcugccuug 77 hsa-miR-103 MIMAT0000101 AGCAGCAUuguacagggcuauga 78 hsa-miR-105 MIMAT0000102 UCAAAUGCucagacuccuguggu 79 hsa-miR-105* MIMAT0004516 ACGGAUGUuugagcaugugcua 80 hsa-miR-106a MIMAT0000103 AAAAGUGCuuacagugcagguag 81 hsa-miR-106a* MIMAT0004517 CUGCAAUGuaagcacuucuuac 82 hsa-miR-107 MIMAT0000104 AGCAGCAUuguacagggcuauca 83 hsa-miR-16-2* MIMAT0004518 CCAAUAUUacugugcugcuuua 84 hsa-miR-192 MIMAT0000222 CUGACCUAugaauugacagcc 85 hsa-miR-192* MIMAT0004543 CUGCCAAUuccauaggucacag 86 hsa-miR-196a MIMAT0000226 UAGGUAGUuucauguuguuggg 87 hsa-miR-197 MIMAT0000227 UUCACCACcuucuccacccagc 88 hsa-miR-198 MIMAT0000228 GGUCCAGAggggagauagguuc 89 hsa-miR-199a-5p MIMAT0000231 CCCAGUGUucagacuaccuguuc 90 hsa-miR-199a-3p MIMAT0000232 ACAGUAGUcugcacauugguua 91 hsa-miR-208a MIMAT0000241 AUAAGACGagcaaaaagcuugu 92 hsa-miR-129-5p MIMAT0000242 CUUUUUGCggucugggcuugc 93 hsa-miR-129* MIMAT0004548 AAGCCCUUaccccaaaaaguau 94 hsa-miR-148a* MIMAT0004549 AAAGUUCUgagacacuccgacu 95 hsa-miR-148a MIMAT0000243 UCAGUGCAcuacagaacuuugu 96 hsa-miR-30c MIMAT0000244 UGUAAACAuccuacacucucagc 97 hsa-miR-30c-2* MIMAT0004550 CUGGGAGAaggcuguuuacucu 98 hsa-miR-30d MIMAT0000245 UGUAAACAuccccgacuggaag 99 hsa-miR-30d* MIMAT0004551 CUUUCAGUcagauguuugcugc 100 hsa-miR-139-5p MIMAT0000250 UCUACAGUgcacgugucuccag 101 hsa-miR-139-3p MIMAT0004552 GGAGACGCggcccuguuggagu 102 hsa-miR-147 MIMAT0000251 GUGUGUGGaaaugcuucugc 103 hsa-miR-7 MIMAT0000252 UGGAAGACuagugauuuuguugu 104 hsa-miR-7-1* MIMAT0004553 CAACAAAUcacagucugccaua 105 hsa-miR-7-2* MIMAT0004554 CAACAAAUcccagucuaccuaa 106 hsa-miR-10a MIMAT0000253 UACCCUGUagauccgaauuugug 107 hsa-miR-10a* MIMAT0004555 CAAAUUCGuaucuaggggaaua 108 hsa-miR-10b MIMAT0000254 UACCCUGUagaaccgaauuugug 109 hsa-miR-10b* MIMAT0004556 ACAGAUUCgauucuaggggaau 110 hsa-miR-34a MIMAT0000255 UGGCAGUGucuuagcugguugu 111 hsa-miR-34a* MIMAT0004557 CAAUCAGCaaguauacugcccu 112 hsa-miR-181a MIMAT0000256 AACAUUCAacgcugucggugagu 113 hsa-miR-181a-2* MIMAT0004558 ACCACUGAccguugacuguacc 114 hsa-miR-181b MIMAT0000257 AACAUUCAuugcugucggugggu 115 hsa-miR-181c MIMAT0000258 AACAUUCAaccugucggugagu 116 hsa-miR-181c* MIMAT0004559 AACCAUCGaccguugaguggac 117 hsa-miR-182 MIMAT0000259 UUUGGCAAugguagaacucacacu 118 hsa-miR-182* MIMAT0000260 UGGUUCUAgacuugccaacua 119 hsa-miR-183 MIMAT0000261 UAUGGCACugguagaauucacu 120 hsa-miR-183* MIMAT0004560 GUGAAUUAccgaagggccauaa 121 hsa-miR-187* MIMAT0004561 GGCUACAAcacaggacccgggc 122 hsa-miR-187 MIMAT0000262 UCGUGUCUuguguugcagccgg 123 hsa-miR-196a* MIMAT0004562 CGGCAACAagaaacugccugag 124 hsa-miR-199b-5p MIMAT0000263 CCCAGUGUuuagacuaucuguuc 125 hsa-miR-199b-3p MIMAT0004563 ACAGUAGUcugcacauugguua 91 hsa-miR-203 MIMAT0000264 GUGAAAUGuuuaggaccacuag 126 hsa-miR-204 MIMAT0000265 UUCCCUUUgucauccuaugccu 127 hsa-miR-205 MIMAT0000266 UCCUUCAUuccaccggagucug 128 hsa-miR-205* MIMAT0009197 GAUUUCAGuggagugaaguuc 129 hsa-miR-210 MIMAT0000267 CUGUGCGUgugacagcggcuga 130 hsa-miR-211 MIMAT0000268 UUCCCUUUgucauccuucgccu 131 hsa-miR-212 MIMAT0000269 UAACAGUCuccagucacggcc 132 hsa-miR-181a* MIMAT0000270 ACCAUCGAccguugauuguacc 133 hsa-miR-214* MIMAT0004564 UGCCUGUCuacacuugcugugc 134 hsa-miR-214 MIMAT0000271 ACAGCAGGcacagacaggcagu 135 hsa-miR-215 MIMAT0000272 AUGACCUAugaauugacagac 136 hsa-miR-216a MIMAT0000273 UAAUCUCAgcuggcaacuguga 137 hsa-miR-217 MIMAT0000274 UACUGCAUcaggaacugauugga 138 hsa-miR-218 MIMAT0000275 UUGUGCUUgaucuaaccaugu 139 hsa-miR-218-1* MIMAT0004565 AUGGUUCCgucaagcaccaugg 140 hsa-miR-218-2* MIMAT0004566 CAUGGUUCugucaagcaccgcg 141 hsa-miR-219-5p MIMAT0000276 UGAUUGUCcaaacgcaauucu 142 hsa-miR-219-1-3p MIMAT0004567 AGAGUUGAgucuggacgucccg 143 hsa-miR-220a MIMAT0000277 CCACACCGuaucugacacuuu 144 hsa-miR-221* MIMAT0004568 ACCUGGCAuacaauguagauuu 145 hsa-miR-221 MIMAT0000278 AGCUACAUugucugcuggguuuc 146 hsa-miR-222* MIMAT0004569 CUCAGUAGccaguguagauccu 147 hsa-miR-222 MIMAT0000279 AGCUACAUcuggcuacugggu 148 hsa-miR-223* MIMAT0004570 CGUGUAUUugacaagcugaguu 149 hsa-miR-223 MIMAT0000280 UGUCAGUUugucaaauacccca 150 hsa-miR-224 MIMAT0000281 CAAGUCACuagugguuccguu 151 hsa-miR-224* MIMAT0009198 AAAAUGGUgcccuagugacuaca 152 hsa-miR-200b* MIMAT0004571 CAUCUUACugggcagcauugga 153 hsa-miR-200b MIMAT0000318 UAAUACUGccugguaaugauga 154 hsa-let-7g MIMAT0000414 UGAGGUAGuaguuuguacaguu 155 hsa-let-7g* MIMAT0004584 CUGUACAGgccacugccuugc 156 hsa-let-7i MIMAT0000415 UGAGGUAGuaguuugugcuguu 157 hsa-let-7i* MIMAT0004585 CUGCGCAAgcuacugccuugcu 158 hsa-miR-1 MIMAT0000416 UGGAAUGUaaagaaguauguau 159 hsa-miR-15b MIMAT0000417 UAGCAGCAcaucaugguuuaca 160 hsa-miR-15b* MIMAT0004586 CGAAUCAUuauuugcugcucua 161 hsa-miR-23b* MIMAT0004587 UGGGUUCCuggcaugcugauuu 162 hsa-miR-23b MIMAT0000418 AUCACAUUgccagggauuacc 163 hsa-miR-27b* MIMAT0004588 AGAGCUUAgcugauuggugaac 164 hsa-miR-27b MIMAT0000419 UUCACAGUggcuaaguucugc 165 hsa-miR-30b MIMAT0000420 UGUAAACAuccuacacucagcu 166 hsa-miR-30b* MIMAT0004589 CUGGGAGGuggauguuuacuuc 167 hsa-miR-122 MIMAT0000421 UGGAGUGUgacaaugguguuug 168 hsa-miR-122* MIMAT0004590 AACGCCAUuaucacacuaaaua 169 hsa-miR-124* MIMAT0004591 CGUGUUCAcagcggaccuugau 170 hsa-miR-124 MIMAT0000422 UAAGGCACgcggugaaugcc 171 hsa-miR-125b MIMAT0000423 UCCCUGAGacccuaacuuguga 172 hsa-miR-125b-1* MIMAT0004592 ACGGGUUAggcucuugggagcu 173 hsa-miR-128 MIMAT0000424 UCACAGUGaaccggucucuuu 174 hsa-miR-130a* MIMAT0004593 UUCACAUUgugcuacugucugc 175 hsa-miR-130a MIMAT0000425 CAGUGCAAuguuaaaagggcau 176 hsa-miR-132* MIMAT0004594 ACCGUGGCuuucgauuguuacu 177 hsa-miR-132 MIMAT0000426 UAACAGUCuacagccauggucg 178 hsa-miR-133a MIMAT0000427 UUUGGUCCccuucaaccagcug 179 hsa-miR-135a MIMAT0000428 UAUGGCUUuuuauuccuauguga 180 hsa-miR-135a* MIMAT0004595 UAUAGGGAuuggagccguggcg 181 hsa-miR-137 MIMAT0000429 UUAUUGCUuaagaauacgcguag 182 hsa-miR-138 MIMAT0000430 AGCUGGUGuugugaaucaggccg 183 hsa-miR-138-2* MIMAT0004596 GCUAUUUCacgacaccaggguu 184 hsa-miR-140-5p MIMAT0000431 CAGUGGUUuuacccuaugguag 185 hsa-miR-140-3p MIMAT0004597 UACCACAGgguagaaccacgg 186 hsa-miR-141* MIMAT0004598 CAUCUUCCaguacaguguugga 187 hsa-miR-141 MIMAT0000432 UAACACUGucugguaaagaugg 188 hsa-miR-142-5p MIMAT0000433 CAUAAAGUagaaagcacuacu 189 hsa-miR-142-3p MIMAT0000434 UGUAGUGUuuccuacuuuaugga 190 hsa-miR-143* MIMAT0004599 GGUGCAGUgcugcaucucuggu 191 hsa-miR-143 MIMAT0000435 UGAGAUGAagcacuguagcuc 192 hsa-miR-144* MIMAT0004600 GGAUAUCAucauauacuguaag 193 hsa-miR-144 MIMAT0000436 UACAGUAUagaugauguacu 194 hsa-miR-145 MIMAT0000437 GUCCAGUUuucccaggaaucccu 195 hsa-miR-145* MIMAT0004601 GGAUUCCUggaaauacuguucu 196 hsa-miR-152 MIMAT0000438 UCAGUGCAugacagaacuugg 197 hsa-miR-153 MIMAT0000439 UUGCAUAGucacaaaagugauc 198 hsa-miR-191 MIMAT0000440 CAACGGAAucccaaaagcagcug 199 hsa-miR-191* MIMAT0001618 GCUGCGCUuggauuucgucccc 200 hsa-miR-9 MIMAT0000441 UCUUUGGUuaucuagcuguauga 201 hsa-miR-9* MIMAT0000442 AUAAAGCUagauaaccgaaagu 202 hsa-miR-125a-5p MIMAT0000443 UCCCUGAGacccuuuaaccuguga 203 hsa-miR-125a-3p MIMAT0004602 ACAGGUGAgguucuugggagcc 204 hsa-miR-125b-2* MIMAT0004603 UCACAAGUcaggcucuugggac 205 hsa-miR-126* MIMAT0000444 CAUUAUUAcuuuugguacgcg 206 hsa-miR-126 MIMAT0000445 UCGUACCGugaguaauaaugcg 207 hsa-miR-127-5p MIMAT0004604 CUGAAGCUcagagggcucugau 208 hsa-miR-127-3p MIMAT0000446 UCGGAUCCgucugagcuuggcu 209 hsa-miR-129-3p MIMAT0004605 AAGCCCUUaccccaaaaagcau 210 hsa-miR-134 MIMAT0000447 UGUGACUGguugaccagagggg 211 hsa-miR-136 MIMAT0000448 ACUCCAUUuguuuugaugaugga 212 hsa-miR-136* MIMAT0004606 CAUCAUCGucucaaaugagucu 213 hsa-miR-138-1* MIMAT0004607 GCUACUUCacaacaccagggcc 214 hsa-miR-146a MIMAT0000449 UGAGAACUgaauuccauggguu 215 hsa-miR-146a* MIMAT0004608 CCUCUGAAauucaguucuucag 216 hsa-miR-149 MIMAT0000450 UCUGGCUCcgugucuucacuccc 217 hsa-miR-149* MIMAT0004609 AGGGAGGGacgggggcugugc 218 hsa-miR-150 MIMAT0000451 UCUCCCAAcccuuguaccagug 219 hsa-miR-150* MIMAT0004610 CUGGUACAggccugggggacag 220 hsa-miR-154 MIMAT0000452 UAGGUUAUccguguugccuucg 221 hsa-miR-154* MIMAT0000453 AAUCAUACacgguugaccuauu 222 hsa-miR-184 MIMAT0000454 UGGACGGAgaacugauaagggu 223 hsa-miR-185 MIMAT0000455 UGGAGAGAaaggcaguuccuga 224 hsa-miR-185* MIMAT0004611 AGGGGCUGgcuuuccucugguc 225 hsa-miR-186 MIMAT0000456 CAAAGAAUucuccuuuugggcu 226 hsa-miR-186* MIMAT0004612 GCCCAAAGgugaauuuuuuggg 227 hsa-miR-188-5p MIMAT0000457 CAUCCCUUgcaugguggaggg 228 hsa-miR-188-3p MIMAT0004613 CUCCCACAugcaggguuugca 229 hsa-miR-190 MIMAT0000458 UGAUAUGUuugauauauuaggu 230 hsa-miR-193a-5p MIMAT0004614 UGGGUCUUugcgggcgagauga 231 hsa-miR-193a-3p MIMAT0000459 AACUGGCCuacaaagucccagu 232 hsa-miR-194 MIMAT0000460 UGUAACAGcaacuccaugugga 233 hsa-miR-195 MIMAT0000461 UAGCAGCAcagaaauauuggc 234 hsa-miR-195* MIMAT0004615 CCAAUAUUggcugugcugcucc 235 hsa-miR-206 MIMAT0000462 UGGAAUGUaaggaagugugugg 236 hsa-miR-320a MIMAT0000510 AAAAGCUGgguugagagggcga 237 hsa-miR-200c* MIMAT0004657 CGUCUUACccagcaguguuugg 238 hsa-miR-200c MIMAT0000617 UAAUACUGccggguaaugaugga 239 hsa-miR-155 MIMAT0000646 UUAAUGCUaaucgugauaggggu 240 hsa-miR-155* MIMAT0004658 CUCCUACAuauuagcauuaaca 241 hsa-miR-194* MIMAT0004671 CCAGUGGGgcugcuguuaucug 242 hsa-miR-106b MIMAT0000680 UAAAGUGCugacagugcagau 243 hsa-miR-106b* MIMAT0004672 CCGCACUGuggguacuugcugc 244 hsa-miR-29c* MIMAT0004673 UGACCGAUuucuccugguguuc 245 hsa-miR-29c MIMAT0000681 UAGCACCAuuugaaaucgguua 246 hsa-miR-30c-1* MIMAT0004674 CUGGGAGAggguuguuuacucc 247 hsa-miR-200a* MIMAT0001620 CAUCUUACcggacagugcugga 248 hsa-miR-200a MIMAT0000682 UAACACUGucugguaacgaugu 249 hsa-miR-302a* MIMAT0000683 ACUUAAACguggauguacuugcu 250 hsa-miR-302a MIMAT0000684 UAAGUGCUuccauguuuugguga 251 hsa-miR-219-2-3p MIMAT0004675 AGAAUUGUggcuggacaucugu 252 hsa-miR-34b* MIMAT0000685 UAGGCAGUgucauuagcugauug 253 hsa-miR-34b MIMAT0004676 CAAUCACUaacuccacugccau 254 hsa-miR-34c-5p MIMAT0000686 AGGCAGUGuaguuagcugauugc 255 hsa-miR-34c-3p MIMAT0004677 AAUCACUAaccacacggccagg 256 hsa-miR-299-5p MIMAT0002890 UGGUUUACcgucccacauacau 257 hsa-miR-299-3p MIMAT0000687 UAUGUGGGaugguaaaccgcuu 258 hsa-miR-301a MIMAT0000688 CAGUGCAAuaguauugucaaagc 259 hsa-miR-99b MIMAT0000689 CACCCGUAgaaccgaccuugcg 260 hsa-miR-99b* MIMAT0004678 CAAGCUCGugucuguggguccg 261 hsa-miR-296-5p MIMAT0000690 AGGGCCCCcccucaauccugu 262 hsa-miR-296-3p MIMAT0004679 GAGGGUUGgguggaggcucucc 263 hsa-miR-130b* MIMAT0004680 ACUCUUUCccuguugcacuac 264 hsa-miR-130b MIMAT0000691 CAGUGCAAugaugaaagggcau 265 hsa-miR-30e MIMAT0000692 UGUAAACAuccuugacuggaag 266 hsa-miR-30e* MIMAT0000693 CUUUCAGUcggauguuuacagc 267 hsa-miR-26a-2* MIMAT0004681 CCUAUUCUugauuacuuguuuc 268 hsa-miR-361-5p MIMAT0000703 UUAUCAGAaucuccagggguac 269 hsa-miR-361-3p MIMAT0004682 UCCCCCAGgugugauucugauuu 270 hsa-miR-362-5p MIMAT0000705 AAUCCUUGgaaccuaggugugagu 271 hsa-miR-362-3p MIMAT0004683 AACACACCuauucaaggauuca 272 hsa-miR-363* MIMAT0003385 CGGGUGGAucacgaugcaauuu 273 hsa-miR-363 MIMAT0000707 AAUUGCACgguauccaucugua 274 hsa-miR-365 MIMAT0000710 UAAUGCCCcuaaaaauccuuau 275 hsa-miR-365* MIMAT0009199 AGGGACUUucaggggcagcugu 276 hsa-miR-302b* MIMAT0000714 ACUUUAACauggaagugcuuuc 277 hsa-miR-302b MIMAT0000715 UAAGUGCUuccauguuuuaguag 278 hsa-miR-302c* MIMAT0000716 UUUAACAUggggguaccugcug 279 hsa-miR-302c MIMAT0000717 UAAGUGCUuccauguuucagugg 280 hsa-miR-302d* MIMAT0004685 ACUUUAACauggaggcacuugc 281 hsa-miR-302d MIMAT0000718 UAAGUGCUuccauguuugagugu 282 hsa-miR-367* MIMAT0004686 ACUGUUGCuaauaugcaacucu 283 hsa-miR-367 MIMAT0000719 AAUUGCACuuuagcaaugguga 284 hsa-miR-376c MIMAT0000720 AACAUAGAggaaauuccacgu 285 hsa-miR-369-5p MIMAT0001621 AGAUCGACcguguuauauucgc 286 hsa-miR-369-3p MIMAT0000721 AAUAAUACaugguugaucuuu 287 hsa-miR-370 MIMAT0000722 GCCUGCUGggguggaaccuggu 288 hsa-miR-371-5p MIMAT0004687 ACUCAAACugugggggcacu 289 hsa-miR-371-3p MIMAT0000723 AAGUGCCGccaucuuuugagugu 290 hsa-miR-372 MIMAT0000724 AAAGUGCUgcgacauuugagcgu 291 hsa-miR-373* MIMAT0000725 ACUCAAAAugggggcgcuuucc 292 hsa-miR-373 MIMAT0000726 GAAGUGCUucgauuuuggggugu 293 hsa-miR-374a MIMAT0000727 UUAUAAUAcaaccugauaagug 294 hsa-miR-374a* MIMAT0004688 CUUAUCAGauuguauuguaauu 295 hsa-miR-375 MIMAT0000728 UUUGUUCGuucggcucgcguga 296 hsa-miR-376a* MIMAT0003386 GUAGAUUCuccuucuaugagua 297 hsa-miR-376a MIMAT0000729 AUCAUAGAggaaaauccacgu 298 hsa-miR-377* MIMAT0004689 AGAGGUUGcccuuggugaauuc 299 hsa-miR-377 MIMAT0000730 AUCACACAaaggcaacuuuugu 300 hsa-miR-378* MIMAT0000731 CUCCUGACuccagguccugugu 301 hsa-miR-378 MIMAT0000732 ACUGGACUuggagucagaagg 302 hsa-miR-379 MIMAT0000733 UGGUAGACuauggaacguagg 303 hsa-miR-379* MIMAT0004690 UAUGUAACaugguccacuaacu 304 hsa-miR-380* MIMAT0000734 UGGUUGACcauagaacaugcgc 305 hsa-miR-380 MIMAT0000735 UAUGUAAUaugguccacaucuu 306 hsa-miR-381 MIMAT0000736 UAUACAAGggcaagcucucugu 307 hsa-miR-382 MIMAT0000737 GAAGUUGUucgugguggauucg 308 hsa-miR-383 MIMAT0000738 AGAUCAGAaggugauuguggcu 309 hsa-miR-340 MIMAT0004692 UUAUAAAGcaaugagacugauu 310 hsa-miR-340* MIMAT0000750 UCCGUCUCaguuacuuuauagc 311 hsa-miR-330-5p MIMAT0004693 UCUCUGGGccugugucuuaggc 312 hsa-miR-330-3p MIMAT0000751 GCAAAGCAcacggccugcagaga 313 hsa-miR-328 MIMAT0000752 CUGGCCCUcucugcccuuccgu 314 hsa-miR-342-5p MIMAT0004694 AGGGGUGCuaucugugauuga 315 hsa-miR-342-3p MIMAT0000753 UCUCACACagaaaucgcacccgu 316 hsa-miR-337-5p MIMAT0004695 GAACGGCUucauacaggaguu 317 hsa-miR-337-3p MIMAT0000754 CUCCUAUAugaugccuuucuuc 318 hsa-miR-323-5p MIMAT0004696 AGGUGGUCcguggcgcguucgc 319 hsa-miR-323-3p MIMAT0000755 CACAUUACacggucgaccucu 320 hsa-miR-326 MIMAT0000756 CCUCUGGGcccuuccuccag 321 hsa-miR-151-5p MIMAT0004697 UCGAGGAGcucacagucuagu 322 hsa-miR-151-3p MIMAT0000757 CUAGACUGaagcuccuugagg 323 hsa-miR-135b MIMAT0000758 UAUGGCUUuucauuccuauguga 324 hsa-miR-135b* MIMAT0004698 AUGUAGGGcuaaaagccauggg 325 hsa-miR-148b* MIMAT0004699 AAGUUCUGuuauacacucaggc 326 hsa-miR-148b MIMAT0000759 UCAGUGCAucacagaacuuugu 327 hsa-miR-331-5p MIMAT0004700 CUAGGUAUggucccagggaucc 328 hsa-miR-331-3p MIMAT0000760 GCCCCUGGgccuauccuagaa 329 hsa-miR-324-5p MIMAT0000761 CGCAUCCCcuagggcauuggugu 330 hsa-miR-324-3p MIMAT0000762 ACUGCCCCaggugcugcugg 331 hsa-miR-338-5p MIMAT0004701 AACAAUAUccuggugcugagug 332 hsa-miR-338-3p MIMAT0000763 UCCAGCAUcagugauuuuguug 333 hsa-miR-339-5p MIMAT0000764 UCCCUGUCcuccaggagcucacg 334 hsa-miR-339-3p MIMAT0004702 UGAGCGCCucgacgacagagccg 335 hsa-miR-335 MIMAT0000765 UCAAGAGCaauaacgaaaaaugu 336 hsa-miR-335* MIMAT0004703 UUUUUCAUuauugcuccugacc 337 hsa-miR-133b MIMAT0000770 UUUGGUCCccuucaaccagcua 338 hsa-miR-325 MIMAT0000771 CCUAGUAGguguccaguaagugu 339 hsa-miR-345 MIMAT0000772 GCUGACUCcuaguccagggcuc 340 hsa-miR-346 MIMAT0000773 UGUCUGCCcgcaugccugccucu 341 hsa-miR-384 MIMAT0001075 AUUCCUAGaaauuguucaua 342 hsa-miR-196b MIMAT0001080 UAGGUAGUuuccuguuguuggg 343 hsa-miR-196b* MIMAT0009201 UCGACAGCacgacacugccuuc 344 hsa-miR-422a MIMAT0001339 ACUGGACUuagggucagaaggc 345 hsa-miR-423-5p MIMAT0004748 UGAGGGGCagagagcgagacuuu 346 hsa-miR-423-3p MIMAT0001340 AGCUCGGUcugaggccccucagu 347 hsa-miR-424 MIMAT0001341 CAGCAGCAauucauguuuugaa 348 hsa-miR-424* MIMAT0004749 CAAAACGUgaggcgcugcuau 349 hsa-miR-425 MIMAT0003393 AAUGACACgaucacucccguuga 350 hsa-miR-425* MIMAT0001343 AUCGGGAAugucguguccgccc 351 hsa-miR-18b MIMAT0001412 UAAGGUGCaucuagugcaguuag 352 hsa-miR-18b* MIMAT0004751 UGCCCUAAaugccccuucuggc 353 hsa-miR-20b MIMAT0001413 CAAAGUGCucauagugcagguag 354 hsa-miR-20b* MIMAT0004752 ACUGUAGUaugggcacuuccag 355 hsa-miR-448 MIMAT0001532 UUGCAUAUguaggaugucccau 356 hsa-miR-429 MIMAT0001536 UAAUACUGucugguaaaaccgu 357 hsa-miR-449a MIMAT0001541 UGGCAGUGuauuguuagcuggu 358 hsa-miR-450a MIMAT0001545 UUUUGCGAuguguuccuaauau 359 hsa-miR-431 MIMAT0001625 UGUCUUGCaggccgucaugca 360 hsa-miR-431* MIMAT0004757 CAGGUCGUcuugcagggcuucu 361 hsa-miR-433 MIMAT0001627 AUCAUGAUgggcuccucggugu 362 hsa-miR-329 MIMAT0001629 AACACACCugguuaaccucuuu 363 hsa-miR-451 MIMAT0001631 AAACCGUUaccauuacugaguu 364 hsa-miR-452 MIMAT0001635 AACUGUUUgcagaggaaacuga 365 hsa-miR-452* MIMAT0001636 CUCAUCUGcaaagaaguaagug 366 hsa-miR-409-5p MIMAT0001638 AGGUUACCcgagcaacuuugcau 367 hsa-miR-409-3p MIMAT0001639 GAAUGUUGcucggugaaccccu 368 hsa-miR-412 MIMAT0002170 ACUUCACCugguccacuagccgu 369 hsa-miR-410 MIMAT0002171 AAUAUAACacagauggccugu 370 hsa-miR-376b MIMAT0002172 AUCAUAGAggaaaauccauguu 371 hsa-miR-483-5p MIMAT0004761 AAGACGGGaggaaagaagggag 372 hsa-miR-483-3p MIMAT0002173 UCACUCCUcuccucccgucuu 373 hsa-miR-484 MIMAT0002174 UCAGGCUCaguccccucccgau 374 hsa-miR-485-5p MIMAT0002175 AGAGGCUGgccgugaugaauuc 375 hsa-miR-485-3p MIMAT0002176 GUCAUACAcggcucuccucucu 376 hsa-miR-486-5p MIMAT0002177 UCCUGUACugagcugccccgag 377 hsa-miR-486-3p MIMAT0004762 CGGGGCAGcucaguacaggau 378 hsa-miR-487a MIMAT0002178 AAUCAUACagggacauccaguu 379 hsa-miR-488* MIMAT0002804 CCCAGAUAauggcacucucaa 380 hsa-miR-488 MIMAT0004763 UUGAAAGGcuauuucuugguc 381 hsa-miR-489 MIMAT0002805 GUGACAUCacauauacggcagc 382 hsa-miR-490-5p MIMAT0004764 CCAUGGAUcuccaggugggu 383 hsa-miR-490-3p MIMAT0002806 CAACCUGGaggacuccaugcug 384 hsa-miR-491-5p MIMAT0002807 AGUGGGGAacccuuccaugagg 385 hsa-miR-491-3p MIMAT0004765 CUUAUGCAagauucccuucuac 386 hsa-miR-511 MIMAT0002808 GUGUCUUUugcucugcaguca 387 hsa-miR-146b-5p MIMAT0002809 UGAGAACUgaauuccauaggcu 388 hsa-miR-146b-3p MIMAT0004766 UGCCCUGUggacucaguucugg 389 hsa-miR-202* MIMAT0002810 UUCCUAUGcauauacuucuuug 390 hsa-miR-202 MIMAT0002811 AGAGGUAUagggcaugggaa 391 hsa-miR-492 MIMAT0002812 AGGACCUGcgggacaagauucuu 392 hsa-miR-493* MIMAT0002813 UUGUACAUgguaggcuuucauu 393 hsa-miR-493 MIMAT0003161 UGAAGGUCuacugugugccagg 394 hsa-miR-432 MIMAT0002814 UCUUGGAGuaggucauugggugg 395 hsa-miR-432* MIMAT0002815 CUGGAUGGcuccuccaugucu 396 hsa-miR-494 MIMAT0002816 UGAAACAUacacgggaaaccuc 397 hsa-miR-495 MIMAT0002817 AAACAAACauggugcacuucuu 398 hsa-miR-496 MIMAT0002818 UGAGUAUUacauggccaaucuc 399 hsa-miR-193b* MIMAT0004767 CGGGGUUUugagggcgagauga 400 hsa-miR-193b MIMAT0002819 AACUGGCCcucaaagucccgcu 401 hsa-miR-497 MIMAT0002820 CAGCAGCAcacugugguuugu 402 hsa-miR-497* MIMAT0004768 CAAACCACacugugguguuaga 403 hsa-miR-181d MIMAT0002821 AACAUUCAuuguugucggugggu 404 hsa-miR-512-5p MIMAT0002822 CACUCAGCcuugagggcacuuuc 405 hsa-miR-512-3p MIMAT0002823 AAGUGCUGucauagcugagguc 406 hsa-miR-498 MIMAT0002824 UUUCAAGCcagggggcguuuuuc 407 hsa-miR-520e MIMAT0002825 AAAGUGCUuccuuuuugaggg 408 hsa-miR-515-5p MIMAT0002826 UUCUCCAAaagaaagcacuuucug 409 hsa-miR-515-3p MIMAT0002827 GAGUGCCUucuuuuggagcguu 410 hsa-miR-519e* MIMAT0002828 UUCUCCAAaagggagcacuuuc 411 hsa-miR-519e MIMAT0002829 AAGUGCCUccuuuuagaguguu 412 hsa-miR-520f MIMAT0002830 AAGUGCUUccuuuuagaggguu 413 hsa-miR-519c-5p MIMAT0002831 CUCUAGAGggaagcgcuuucug 414 hsa-miR-519c-3p MIMAT0002832 AAAGUGCAucuuuuuagaggau 415 hsa-miR-520a-5p MIMAT0002833 CUCCAGAGggaaguacuuucu 416 hsa-miR-520a-3p MIMAT0002834 AAAGUGCUucccuuuggacugu 417 hsa-miR-526b MIMAT0002835 CUCUUGAGggaagcacuuucugu 418 hsa-miR-526b* MIMAT0002836 GAAAGUGCuuccuuuuagaggc 419 hsa-miR-519b-5p MIMAT0005454 CUCUAGAGggaagcgcuuucug 414 hsa-miR-519b-3p MIMAT0002837 AAAGUGCAuccuuuuagagguu 420 hsa-miR-525-5p MIMAT0002838 CUCCAGAGggaugcacuuucu 421 hsa-miR-525-3p MIMAT0002839 GAAGGCGCuucccuuuagagcg 422 hsa-miR-523* MIMAT0005449 CUCUAGAGggaagcgcuuucug 414 hsa-miR-523 MIMAT0002840 GAACGCGCuucccuauagagggu 423 hsa-miR-518f* MIMAT0002841 CUCUAGAGggaagcacuuucuc 424 hsa-miR-518f MIMAT0002842 GAAAGCGCuucucuuuagagg 425 hsa-miR-520b MIMAT0002843 AAAGUGCUuccuuuuagaggg 426 hsa-miR-518b MIMAT0002844 CAAAGCGCuccccuuuagaggu 427 hsa-miR-526a MIMAT0002845 CUCUAGAGggaagcacuuucug 428 hsa-miR-520c-5p MIMAT0005455 CUCUAGAGggaagcacuuucug 428 hsa-miR-520c-3p MIMAT0002846 AAAGUGCUuccuuuuagagggu 429 hsa-miR-518c* MIMAT0002847 UCUCUGGAgggaagcacuuucug 430 hsa-miR-518c MIMAT0002848 CAAAGCGCuucucuuuagagugu 431 hsa-miR-524-5p MIMAT0002849 CUACAAAGggaagcacuuucuc 432 hsa-miR-524-3p MIMAT0002850 GAAGGCGCuucccuuuggagu 433 hsa-miR-517* MIMAT0002851 CCUCUAGAuggaagcacugucu 434 hsa-miR-517a MIMAT0002852 AUCGUGCAucccuuuagagugu 435 hsa-miR-519d MIMAT0002853 CAAAGUGCcucccuuuagagug 436 hsa-miR-521 MIMAT0002854 AACGCACUucccuuuagagugu 437 hsa-miR-520d-5p MIMAT0002855 CUACAAAGggaagcccuuuc 438 hsa-miR-520d-3p MIMAT0002856 AAAGUGCUucucuuuggugggu 439 hsa-miR-517b MIMAT0002857 UCGUGCAUcccuuuagaguguu 440 hsa-miR-520g MIMAT0002858 ACAAAGUGcuucccuuuagagugu 441 hsa-miR-516b MIMAT0002859 AUCUGGAGguaagaagcacuuu 442 hsa-miR-516b* MIMAT0002860 UGCUUCCUuucagagggu 443 hsa-miR-518e* MIMAT0005450 CUCUAGAGggaagcgcuuucug 414 hsa-miR-518e MIMAT0002861 AAAGCGCUucccuucagagug 444 hsa-miR-518a-5p MIMAT0005457 CUGCAAAGggaagcccuuuc 445 hsa-miR-518a-3p MIMAT0002863 GAAAGCGCuucccuuugcugga 446 hsa-miR-518d-5p MIMAT0005456 CUCUAGAGggaagcacuuucug 428 hsa-miR-518d-3p MIMAT0002864 CAAAGCGCuucccuuuggagc 447 hsa-miR-517c MIMAT0002866 AUCGUGCAuccuuuuagagugu 448 hsa-miR-520h MIMAT0002867 ACAAAGUGcuucccuuuagagu 449 hsa-miR-522* MIMAT0005451 CUCUAGAGggaagcgcuuucug 414 hsa-miR-522 MIMAT0002868 AAAAUGGUucccuuuagagugu 450 hsa-miR-519a* MIMAT0005452 CUCUAGAGggaagcgcuuucug 414 hsa-miR-519a MIMAT0002869 AAAGUGCAuccuuuuagagugu 451 hsa-miR-527 MIMAT0002862 CUGCAAAGggaagcccuuuc 445 hsa-miR-516a-5p MIMAT0004770 UUCUCGAGgaaagaagcacuuuc 452 hsa-miR-516a-3p MIMAT0006778 UGCUUCCUuucagagggu 443 hsa-miR-499-5p MIMAT0002870 UUAAGACUugcagugauguuu 453 hsa-miR-499-3p MIMAT0004772 AACAUCACagcaagucugugcu 454 hsa-miR-500 MIMAT0004773 UAAUCCUUgcuaccugggugaga 455 hsa-miR-500* MIMAT0002871 AUGCACCUgggcaaggauucug 456 hsa-miR-501-5p MIMAT0002872 AAUCCUUUgucccugggugaga 457 hsa-miR-501-3p MIMAT0004774 AAUGCACCcgggcaaggauucu 458 hsa-miR-502-5p MIMAT0002873 AUCCUUGCuaucugggugcua 459 hsa-miR-502-3p MIMAT0004775 AAUGCACCugggcaaggauuca 460 hsa-miR-503 MIMAT0002874 UAGCAGCGggaacaguucugcag 461 hsa-miR-504 MIMAT0002875 AGACCCUGgucugcacucuauc 462 hsa-miR-505* MIMAT0004776 GGGAGCCAggaaguauugaugu 463 hsa-miR-505 MIMAT0002876 CGUCAACAcuugcugguuuccu 464 hsa-miR-513a-5p MIMAT0002877 UUCACAGGgaggugucau 465 hsa-miR-513a-3p MIMAT0004777 UAAAUUUCaccuuucugagaagg 466 hsa-miR-506 MIMAT0002878 UAAGGCACccuucugaguaga 467 hsa-miR-507 MIMAT0002879 UUUUGCACcuuuuggagugaa 468 hsa-miR-508-5p MIMAT0004778 UACUCCAGagggcgucacucaug 469 hsa-miR-508-3p MIMAT0002880 UGAUUGUAgccuuuuggaguaga 470 hsa-miR-509-5p MIMAT0004779 UACUGCAGacaguggcaauca 471 hsa-miR-509-3p MIMAT0002881 UGAUUGGUacgucuguggguag 472 hsa-miR-510 MIMAT0002882 UACUCAGGagaguggcaaucac 473 hsa-miR-514 MIMAT0002883 AUUGACACuucugugaguaga 474 hsa-miR-532-5p MIMAT0002888 CAUGCCUUgaguguaggaccgu 475 hsa-miR-532-3p MIMAT0004780 CCUCCCACacccaaggcuugca 476 hsa-miR-455-5p MIMAT0003150 UAUGUGCCuuuggacuacaucg 477 hsa-miR-455-3p MIMAT0004784 GCAGUCCAugggcauauacac 478 hsa-miR-539 MIMAT0003163 GGAGAAAUuauccuuggugugu 479 hsa-miR-544 MIMAT0003164 AUUCUGCAuuuuuagcaaguuc 480 hsa-miR-545* MIMAT0004785 UCAGUAAAuguuuauuagauga 481 hsa-miR-545 MIMAT0003165 UCAGCAAAcauuuauugugugc 482 hsa-miR-487b MIMAT0003180 AAUCGUACagggucauccacuu 483 hsa-miR-551a MIMAT0003214 GCGACCCAcucuugguuucca 484 hsa-miR-552 MIMAT0003215 AACAGGUGacugguuagacaa 485 hsa-miR-553 MIMAT0003216 AAAACGGUgagauuuuguuuu 486 hsa-miR-554 MIMAT0003217 GCUAGUCCugacucagccagu 487 hsa-miR-92b* MIMAT0004792 AGGGACGGgacgcggugcagug 488 hsa-miR-92b MIMAT0003218 UAUUGCACucgucccggccucc 489 hsa-miR-555 MIMAT0003219 AGGGUAAGcugaaccucugau 490 hsa-miR-556-5p MIMAT0003220 GAUGAGCUcauuguaauaugag 491 hsa-miR-556-3p MIMAT0004793 AUAUUACCauuagcucaucuuu 492 hsa-miR-557 MIMAT0003221 GUUUGCACgggugggccuugucu 493 hsa-miR-558 MIMAT0003222 UGAGCUGCuguaccaaaau 494 hsa-miR-559 MIMAT0003223 UAAAGUAAauaugcaccaaaa 495 hsa-miR-561 MIMAT0003225 CAAAGUUUaagauccuugaagu 496 hsa-miR-562 MIMAT0003226 AAAGUAGCuguaccauuugc 497 hsa-miR-563 MIMAT0003227 AGGUUGACauacguuuccc 498 hsa-miR-564 MIMAT0003228 AGGCACGGugucagcaggc 499 hsa-miR-566 MIMAT0003230 GGGCGCCUgugaucccaac 500 hsa-miR-567 MIMAT0003231 AGUAUGUUcuuccaggacagaac 501 hsa-miR-568 MIMAT0003232 AUGUAUAAauguauacacac 502 hsa-miR-552b* MIMAT0004794 GAAAUCAAgcgugggugagacc 503 hsa-miR-551b MIMAT0003233 GCGACCCAuacuugguuucag 504 hsa-miR-569 MIMAT0003234 AGUUAAUGaauccuggaaagu 505 hsa-miR-570 MIMAT0003235 CGAAAACAgcaauuaccuuugc 506 hsa-miR-571 MIMAT0003236 UGAGUUGGccaucugagugag 507 hsa-miR-572 MIMAT0003237 GUCCGCUCggcgguggccca 508 hsa-miR-573 MIMAT0003238 CUGAAGUGauguguaacugaucag 509 hsa-miR-574-5p MIMAT0004795 UGAGUGUGugugugugagugugu 510 hsa-miR-574-3p MIMAT0003239 CACGCUCAugcacacacccaca 511 hsa-miR-575 MIMAT0003240 GAGCCAGUuggacaggagc 512 hsa-miR-576-5p MIMAT0003241 AUUCUAAUuucuccacgucuuu 513 hsa-miR-576-3p MIMAT0004796 AAGAUGUGgaaaaauuggaauc 514 hsa-miR-577 MIMAT0003242 UAGAUAAAauauugguaccug 515 hsa-miR-578 MIMAT0003243 CUUCUUGUgcucuaggauugu 516 hsa-miR-579 MIMAT0003244 UUCAUUUGguauaaaccgcgauu 517 hsa-miR-580 MIMAT0003245 UUGAGAAUgaugaaucauuagg 518 hsa-miR-581 MIMAT0003246 UCUUGUGUucucuagaucagu 519 hsa-miR-582-5p MIMAT0003247 UUACAGUUguucaaccaguuacu 520 hsa-miR-582-3p MIMAT0004797 UAACUGGUugaacaacugaacc 521 hsa-miR-583 MIMAT0003248 CAAAGAGGaaggucccauuac 522 hsa-miR-584 MIMAT0003249 UUAUGGUUugccugggacugag 523 hsa-miR-585 MIMAT0003250 UGGGCGUAucuguaugcua 524 hsa-miR-548a-3p MIMAT0003251 CAAAACUGgcaauuacuuuugc 525 hsa-miR-586 MIMAT0003252 UAUGCAUUguauuuuuaggucc 526 hsa-miR-587 MIMAT0003253 UUUCCAUAggugaugagucac 527 hsa-miR-548b-5p MIMAT0004798 AAAAGUAAuugugguuuuggcc 528 hsa-miR-548b-3p MIMAT0003254 CAAGAACCucaguugcuuuugu 529 hsa-miR-588 MIMAT0003255 UUGGCCACaauggguuagaac 530 hsa-miR-589 MIMAT0004799 UGAGAACCacgucugcucugag 531 hsa-miR-589* MIMAT0003256 UCAGAACAaaugccgguucccaga 532 hsa-miR-550 MIMAT0004800 AGUGCCUGagggaguaagagccc 533 hsa-miR-550* MIMAT0003257 UGUCUUACucccucaggcacau 534 hsa-miR-590-5p MIMAT0003258 GAGCUUAUucauaaaagugcag 535 hsa-miR-590-3p MIMAT0004801 UAAUUUUAuguauaagcuagu 536 hsa-miR-591 MIMAT0003259 AGACCAUGgguucucauugu 537 hsa-miR-592 MIMAT0003260 UUGUGUCAauaugcgaugaugu 538 hsa-miR-593* MIMAT0003261 AGGCACCAgccaggcauugcucagc 539 hsa-miR-593 MIMAT0004802 UGUCUCUGcugggguuucu 540 hsa-miR-595 MIMAT0003263 GAAGUGUGccguggugugucu 541 hsa-miR-596 MIMAT0003264 AAGCCUGCccggcuccucggg 542 hsa-miR-597 MIMAT0003265 UGUGUCACucgaugaccacugu 543 hsa-miR-598 MIMAT0003266 UACGUCAUcguugucaucguca 544 hsa-miR-599 MIMAT0003267 GUUGUGUCaguuuaucaaac 545 hsa-miR-548a-5p MIMAT0004803 AAAAGUAAuugcgaguuuuacc 546 hsa-miR-600 MIMAT0003268 ACUUACAGacaagagccuugcuc 547 hsa-miR-601 MIMAT0003269 UGGUCUAGgauuguuggaggag 548 hsa-miR-602 MIMAT0003270 GACACGGGcgacagcugcggccc 549 hsa-miR-603 MIMAT0003271 CACACACUgcaauuacuuuugc 550 hsa-miR-604 MIMAT0003272 AGGCUGCGgaauucaggac 551 hsa-miR-605 MIMAT0003273 UAAAUCCCauggugccuucuccu 552 hsa-miR-606 MIMAT0003274 AAACUACUgaaaaucaaagau 553 hsa-miR-607 MIMAT0003275 GUUCAAAUccagaucuauaac 554 hsa-miR-608 MIMAT0003276 AGGGGUGGuguugggacagcuccgu 555 hsa-miR-609 MIMAT0003277 AGGGUGUUucucucaucucu 556 hsa-miR-610 MIMAT0003278 UGAGCUAAaugugugcuggga 557 hsa-miR-611 MIMAT0003279 GCGAGGACcccucggggucugac 558 hsa-miR-612 MIMAT0003280 GCUGGGCAgggcuucugagcuccuu 559 hsa-miR-613 MIMAT0003281 AGGAAUGUuccuucuuugcc 560 hsa-miR-614 MIMAT0003282 GAACGCCUguucuugccaggugg 561 hsa-miR-615-5p MIMAT0004804 GGGGGUCCccggugcucggauc 562 hsa-miR-615-3p MIMAT0003283 UCCGAGCCugggucucccucuu 563 hsa-miR-616* MIMAT0003284 ACUCAAAAcccuucagugacuu 564 hsa-miR-616 MIMAT0004805 AGUCAUUGgaggguuugagcag 565 hsa-miR-548c-5p MIMAT0004806 AAAAGUAAuugcgguuuuugcc 566 hsa-miR-548c-3p MIMAT0003285 CAAAAAUCucaauuacuuuugc 567 hsa-miR-617 MIMAT0003286 AGACUUCCcauuugaagguggc 568 hsa-miR-618 MIMAT0003287 AAACUCUAcuuguccuucugagu 569 hsa-miR-619 MIMAT0003288 GACCUGGAcauguuugugcccagu 570 hsa-miR-620 MIMAT0003289 AUGGAGAUagauauagaaau 571 hsa-miR-621 MIMAT0003290 GGCUAGCAacagcgcuuaccu 572 hsa-miR-622 MIMAT0003291 ACAGUCUGcugagguuggagc 573 hsa-miR-623 MIMAT0003292 AUCCCUUGcaggggcuguugggu 574 hsa-miR-624* MIMAT0003293 UAGUACCAguaccuuguguuca 575 hsa-miR-624 MIMAT0004807 CACAAGGUauugguauuaccu 576 hsa-miR-625 MIMAT0003294 AGGGGGAAaguucuauagucc 577 hsa-miR-625* MIMAT0004808 GACUAUAGaacuuucccccuca 578 hsa-miR-626 MIMAT0003295 AGCUGUCUgaaaaugucuu 579 hsa-miR-627 MIMAT0003296 GUGAGUCUcuaagaaaagagga 580 hsa-miR-628-5p MIMAT0004809 AUGCUGACauauuuacuagagg 581 hsa-miR-628-3p MIMAT0003297 UCUAGUAAgaguggcagucga 582 hsa-miR-629 MIMAT0004810 UGGGUUUAcguugggagaacu 583 hsa-miR-629* MIMAT0003298 GUUCUCCCaacguaagcccagc 584 hsa-miR-630 MIMAT0003299 AGUAUUCUguaccagggaaggu 585 hsa-miR-631 MIMAT0003300 AGACCUGGcccagaccucagc 586 hsa-miR-33b MIMAT0003301 GUGCAUUGcuguugcauugc 587 hsa-miR-33b* MIMAT0004811 CAGUGCCUcggcagugcagccc 588 hsa-miR-632 MIMAT0003302 GUGUCUGCuuccuguggga 589 hsa-miR-633 MIMAT0003303 CUAAUAGUaucuaccacaauaaa 590 hsa-miR-634 MIMAT0003304 AACCAGCAccccaacuuuggac 591 hsa-miR-635 MIMAT0003305 ACUUGGGCacugaaacaaugucc 592 hsa-miR-636 MIMAT0003306 UGUGCUUGcucgucccgcccgca 593 hsa-miR-637 MIMAT0003307 ACUGGGGGcuuucgggcucugcgu 594 hsa-miR-638 MIMAT0003308 AGGGAUCGcgggcggguggcggccu 595 hsa-miR-639 MIMAT0003309 AUCGCUGCgguugcgagcgcugu 596 hsa-miR-640 MIMAT0003310 AUGAUCCAggaaccugccucu 597 hsa-miR-641 MIMAT0003311 AAAGACAUaggauagagucaccuc 598 hsa-miR-642 MIMAT0003312 GUCCCUCUccaaaugugucuug 599 hsa-miR-643 MIMAT0003313 ACUUGUAUgcuagcucagguag 600 hsa-miR-644 MIMAT0003314 AGUGUGGCuuucuuagagc 601 hsa-miR-645 MIMAT0003315 UCUAGGCUgguacugcuga 602 hsa-miR-646 MIMAT0003316 AAGCAGCUgccucugaggc 603 hsa-miR-647 MIMAT0003317 GUGGCUGCacucacuuccuuc 604 hsa-miR-648 MIMAT0003318 AAGUGUGCagggcacuggu 605 hsa-miR-649 MIMAT0003319 AAACCUGUguuguucaagaguc 606 hsa-miR-650 MIMAT0003320 AGGAGGCAgcgcucucaggac 607 hsa-miR-651 MIMAT0003321 UUUAGGAUaagcuugacuuuug 608 hsa-miR-652 MIMAT0003322 AAUGGCGCcacuaggguugug 609 hsa-miR-548d-5p MIMAT0004812 AAAAGUAAuugugguuuuugcc 610 hsa-miR-548d-3p MIMAT0003323 CAAAAACCacaguuucuuuugc 611 hsa-miR-661 MIMAT0003324 UGCCUGGGucucuggccugcgcgu 612 hsa-miR-662 MIMAT0003325 UCCCACGUuguggcccagcag 613 hsa-miR-663 MIMAT0003326 AGGCGGGGcgccgcgggaccgc 614 hsa-miR-449b MIMAT0003327 AGGCAGUGuauuguuagcuggc 615 hsa-miR-449b* MIMAT0009203 CAGCCACAacuacccugccacu 616 hsa-miR-653 MIMAT0003328 GUGUUGAAacaaucucuacug 617 hsa-miR-411 MIMAT0003329 UAGUAGACcguauagcguacg 618 hsa-miR-411* MIMAT0004813 UAUGUAACacgguccacuaacc 619 hsa-miR-654-5p MIMAT0003330 UGGUGGGCcgcagaacaugugc 620 hsa-miR-654-3p MIMAT0004814 UAUGUCUGcugaccaucaccuu 621 hsa-miR-655 MIMAT0003331 AUAAUACAugguuaaccucuuu 622 hsa-miR-656 MIMAT0003332 AAUAUUAUacagucaaccucu 623 hsa-miR-549 MIMAT0003333 UGACAACUauggaugagcucu 624 hsa-miR-657 MIMAT0003335 GGCAGGUUcucacccucucuagg 625 hsa-miR-658 MIMAT0003336 GGCGGAGGgaaguagguccguuggu 626 hsa-miR-659 MIMAT0003337 CUUGGUUCagggagggucccca 627 hsa-miR-660 MIMAT0003338 UACCCAUUgcauaucggaguug 628 hsa-miR-421 MIMAT0003339 AUCAACAGacauuaauugggcgc 629 hsa-miR-542-5p MIMAT0003340 UCGGGGAUcaucaugucacgaga 630 hsa-miR-542-3p MIMAT0003389 UGUGACAGauugauaacugaaa 631 hsa-miR-758 MIMAT0003879 UUUGUGACcugguccacuaacc 632 hsa-miR-1264 MIMAT0005791 CAAGUCUUauuugagcaccuguu 633 hsa-miR-671-5p MIMAT0003880 AGGAAGCCcuggaggggcuggag 634 hsa-miR-671-3p MIMAT0004819 UCCGGUUCucagggcuccacc 635 hsa-miR-668 MIMAT0003881 UGUCACUCggcucggcccacuac 636 hsa-miR-767-5p MIMAT0003882 UGCACCAUgguugucugagcaug 637 hsa-miR-767-3p MIMAT0003883 UCUGCUCAuaccccaugguuucu 638 hsa-miR-1224-5p MIMAT0005458 GUGAGGACucgggaggugg 639 hsa-miR-1224-3p MIMAT0005459 CCCCACCUccucucuccucag 640 hsa-miR-320b MIMAT0005792 AAAAGCUGgguugagagggcaa 641 hsa-miR-320c MIMAT0005793 AAAAGCUGgguugagagggu 642 hsa-miR-1296 MIMAT0005794 UUAGGGCCcuggcuccaucucc 643 hsa-miR-1468 MIMAT0006789 CUCCGUUUgccuguuucgcug 644 hsa-miR-1323 MIMAT0005795 UCAAAACUgaggggcauuuucu 645 hsa-miR-1271 MIMAT0005796 CUUGGCACcuagcaagcacuca 646 hsa-miR-1301 MIMAT0005797 UUGCAGCUgccugggagugacuuc 647 hsa-miR-454* MIMAT0003884 ACCCUAUCaauauugucucugc 648 hsa-miR-454 MIMAT0003885 UAGUGCAAuauugcuuauagggu 649 hsa-miR-1185 MIMAT0005798 AGAGGAUAcccuuuguauguu 650 hsa-miR-449c MIMAT0010251 UAGGCAGUguauugcuagcggcugu 651 hsa-miR-449c* MIMAT0013771 UUGCUAGUugcacuccucucugu 652 hsa-miR-1283 MIMAT0005799 UCUACAAAggaaagcgcuuucu 653 hsa-miR-769-5p MIMAT0003886 UGAGACCUcuggguucugagcu 654 hsa-miR-769-3p MIMAT0003887 CUGGGAUCuccggggucuugguu 655 hsa-miR-766 MIMAT0003888 ACUCCAGCcccacagccucagc 656 hsa-miR-762 MIMAT0010313 GGGGCUGGggccggggccgagc 657 hsa-miR-802 MIMAT0004185 CAGUAACAaagauucauccuugu 658 hsa-miR-670 MIMAT0010357 GUCCCUGAguguauguggug 659 hsa-miR-1298 MIMAT0005800 UUCAUUCGgcuguccagaugua 660 hsa-miR-2113 MIMAT0009206 AUUUGUGCuuggcucugucac 661 hsa-miR-761 MIMAT0010364 GCAGCAGGgugaaacugacaca 662 hsa-miR-764 MIMAT0010367 GCAGGUGCucacuuguccuccu 663 hsa-miR-759 MIMAT0010497 GCAGAGUGcaaacaauuuugac 664 hsa-miR-765 MIMAT0003945 UGGAGGAGaaggaaggugaug 665 hsa-miR-770-5p MIMAT0003948 UCCAGUACcacgugucagggcca 666 hsa-miR-675 MIMAT0004284 UGGUGCGGagagggcccacagug 667 hsa-miR-675* MIMAT0006790 CUGUAUGCccucaccgcuca 668 hsa-miR-298 MIMAT0004901 AGCAGAAGcagggagguucuccca 669 hsa-miR-891a MIMAT0004902 UGCAACGAaccugagccacuga 670 hsa-miR-300 MIMAT0004903 UAUACAAGggcagacucucucu 671 hsa-miR-886-5p MIMAT0004905 CGGGUCGGaguuagcucaagcgg 672 hsa-miR-886-3p MIMAT0004906 CGCGGGUGcuuacugacccuu 673 hsa-miR-892a MIMAT0004907 CACUGUGUccuuucugcguag 674 hsa-miR-220b MIMAT0004908 CCACCACCgugucugacacuu 675 hsa-miR-450b-5p MIMAT0004909 UUUUGCAAuauguuccugaaua 676 hsa-miR-450b-3p MIMAT0004910 UUGGGAUCauuuugcauccaua 677 hsa-miR-874 MIMAT0004911 CUGCCCUGgcccgagggaccga 678 hsa-miR-890 MIMAT0004912 UACUUGGAaaggcaucaguug 679 hsa-miR-891b MIMAT0004913 UGCAACUUaccugagucauuga 680 hsa-miR-220c MIMAT0004915 ACACAGGGcuguugugaagacu 681 hsa-miR-888 MIMAT0004916 UACUCAAAaagcugucaguca 682 hsa-miR-888* MIMAT0004917 GACUGACAccucuuugggugaa 683 hsa-miR-892b MIMAT0004918 CACUGGCUccuuucuggguaga 684 hsa-miR-541* MIMAT0004919 AAAGGAUUcugcugucggucccacu 685 hsa-miR-541 MIMAT0004920 UGGUGGGCacagaaucuggacu 686 hsa-miR-889 MIMAT0004921 UUAAUAUCggacaaccauugu 687 hsa-miR-875-5p MIMAT0004922 UAUACCUCaguuuuaucaggug 688 hsa-miR-875-3p MIMAT0004923 CCUGGAAAcacugagguugug 689 hsa-miR-876-5p MIMAT0004924 UGGAUUUCuuugugaaucacca 690 hsa-miR-876-3p MIMAT0004925 UGGUGGUUuacaaaguaauuca 691 hsa-miR-708 MIMAT0004926 AAGGAGCUuacaaucuagcuggg 692 hsa-miR-708* MIMAT0004927 CAACUAGAcugugagcuucuag 693 hsa-miR-147b MIMAT0004928 GUGUGCGGaaaugcuucugcua 694 hsa-miR-190b MIMAT0004929 UGAUAUGUuugauauuggguu 695 hsa-miR-744 MIMAT0004945 UGCGGGGCuagggcuaacagca 696 hsa-miR-744* MIMAT0004946 CUGUUGCCacuaaccucaaccu 697 hsa-miR-885-5p MIMAT0004947 UCCAUUACacuacccugccucu 698 hsa-miR-885-3p MIMAT0004948 AGGCAGCGggguguaguggaua 699 hsa-miR-877 MIMAT0004949 GUAGAGGAgauggcgcaggg 700 hsa-miR-877* MIMAT0004950 UCCUCUUCucccuccucccag 701 hsa-miR-887 MIMAT0004951 GUGAACGGgcgccaucccgagg 702 hsa-miR-665 MIMAT0004952 ACCAGGAGgcugaggccccu 703 hsa-miR-873 MIMAT0004953 GCAGGAACuugugagucuccu 704 hsa-miR-543 MIMAT0004954 AAACAUUCgcggugcacuucuu 705 hsa-miR-374b MIMAT0004955 AUAUAAUAcaaccugcuaagug 706 hsa-miR-374b* MIMAT0004956 CUUAGCAGguuguauuaucauu 707 hsa-miR-760 MIMAT0004957 CGGCUCUGggucugugggga 708 hsa-miR-301b MIMAT0004958 CAGUGCAAugauauugucaaagc 709 hsa-miR-216b MIMAT0004959 AAAUCUCUgcaggcaaauguga 710 hsa-miR-208b MIMAT0004960 AUAAGACGaacaaaagguuugu 711 hsa-miR-920 MIMAT0004970 GGGGAGCUguggaagcagua 712 hsa-miR-921 MIMAT0004971 CUAGUGAGggacagaaccaggauuc 713 hsa-miR-922 MIMAT0004972 GCAGCAGAgaauaggacuacguc 714 hsa-miR-924 MIMAT0004974 AGAGUCUUgugaugucuugc 715 hsa-miR-509-3-5p MIMAT0004975 UACUGCAGacguggcaaucaug 716 hsa-miR-933 MIMAT0004976 UGUGCGCAgggagaccucuccc 717 hsa-miR-934 MIMAT0004977 UGUCUACUacuggagacacugg 718 hsa-miR-935 MIMAT0004978 CCAGUUACcgcuuccgcuaccgc 719 hsa-miR-936 MIMAT0004979 ACAGUAGAgggaggaaucgcag 720 hsa-miR-937 MIMAT0004980 AUCCGCGCucugacucucugcc 721 hsa-miR-938 MIMAT0004981 UGCCCUUAaaggugaacccagu 722 hsa-miR-939 MIMAT0004982 UGGGGAGCugaggcucugggggug 723 hsa-miR-940 MIMAT0004983 AAGGCAGGgcccccgcucccc 724 hsa-miR-941 MIMAT0004984 CACCCGGCugugugcacaugugc 725 hsa-miR-942 MIMAT0004985 UCUUCUCUguuuuggccaugug 726 hsa-miR-943 MIMAT0004986 CUGACUGUugccguccuccag 727 hsa-miR-944 MIMAT0004987 AAAUUAUUguacaucggaugag 728 hsa-miR-297 MIMAT0004450 AUGUAUGUgugcaugugcaug 729 hsa-miR-1178 MIMAT0005823 UUGCUCACuguucuucccuag 730 hsa-miR-1179 MIMAT0005824 AAGCAUUCuuucauugguugg 731 hsa-miR-1180 MIMAT0005825 UUUCCGGCucgcgugggugugu 732 hsa-miR-1181 MIMAT0005826 CCGUCGCCgccacccgagccg 733 hsa-miR-1182 MIMAT0005827 GAGGGUCUugggagggaugugac 734 hsa-miR-1183 MIMAT0005828 CACUGUAGgugauggugagagugggca 735 hsa-miR-1184 MIMAT0005829 CCUGCAGCgacuugauggcuucc 736 hsa-miR-1225-5p MIMAT0005572 GUGGGUACggcccagugggggg 737 hsa-miR-1225-3p MIMAT0005573 UGAGCCCCugugccgcccccag 738 hsa-miR-1226* MIMAT0005576 GUGAGGGCaugcaggccuggaugggg 739 hsa-miR-1226 MIMAT0005577 UCACCAGCccuguguucccuag 740 hsa-miR-1227 MIMAT0005580 CGUGCCACccuuuuccccag 741 hsa-miR-1228* MIMAT0005582 GUGGGCGGgggcaggugugug 742 hsa-miR-1228 MIMAT0005583 UCACACCUgccucgcccccc 743 hsa-miR-1229 MIMAT0005584 CUCUCACCacugcccucccacag 744 hsa-miR-1231 MIMAT0005586 GUGUCUGGgcggacagcugc 745 hsa-miR-1233 MIMAT0005588 UGAGCCCUguccucccgcag 746 hsa-miR-1234 MIMAT0005589 UCGGCCUGaccacccaccccac 747 hsa-miR-1236 MIMAT0005591 CCUCUUCCccuugucucuccag 748 hsa-miR-1237 MIMAT0005592 UCCUUCUGcuccgucccccag 749 hsa-miR-1238 MIMAT0005593 CUUCCUCGucugucugcccc 750 hsa-miR-1200 MIMAT0005863 CUCCUGAGccauucugagccuc 751 hsa-miR-1201 MIMAT0005864 AGCCUGAUuaaacacaugcucuga 752 hsa-miR-1202 MIMAT0005865 GUGCCAGCugcagugggggag 753 hsa-miR-1203 MIMAT0005866 CCCGGAGCcaggaugcagcuc 754 hsa-miR-663b MIMAT0005867 GGUGGCCCggccgugccugagg 755 hsa-miR-1204 MIMAT0005868 UCGUGGCCuggucuccauuau 756 hsa-miR-1205 MIMAT0005869 UCUGCAGGguuugcuuugag 757 hsa-miR-1206 MIMAT0005870 UGUUCAUGuagauguuuaagc 758 hsa-miR-1207-5p MIMAT0005871 UGGCAGGGaggcugggagggg 759 hsa-miR-1207-3p MIMAT0005872 UCAGCUGGcccucauuuc 760 hsa-miR-1208 MIMAT0005873 UCACUGUUcagacaggcgga 761 hsa-miR-548e MIMAT0005874 AAAAACUGagacuacuuuugca 762 hsa-miR-548j MIMAT0005875 AAAAGUAAuugcggucuuuggu 763 hsa-miR-1285 MIMAT0005876 UCUGGGCAacaaagugagaccu 764 hsa-miR-1286 MIMAT0005877 UGCAGGACcaagaugagcccu 765 hsa-miR-1287 MIMAT0005878 UGCUGGAUcagugguucgaguc 766 hsa-miR-1289 MIMAT0005879 UGGAGUCCaggaaucugcauuuu 767 hsa-miR-1290 MIMAT0005880 UGGAUUUUuggaucaggga 768 hsa-miR-1291 MIMAT0005881 UGGCCCUGacugaagaccagcagu 769 hsa-miR-548k MIMAT0005882 AAAAGUACuugcggauuuugcu 770 hsa-miR-1293 MIMAT0005883 UGGGUGGUcuggagauuugugc 771 hsa-miR-1294 MIMAT0005884 UGUGAGGUuggcauuguugucu 772 hsa-miR-1295 MIMAT0005885 UUAGGCCGcagaucuggguga 773 hsa-miR-1297 MIMAT0005886 UUCAAGUAauucaggug 774 hsa-miR-1299 MIMAT0005887 UUCUGGAAuucugugugaggga 775 hsa-miR-5481 MIMAT0005889 AAAAGUAUuugcggguuuuguc 776 hsa-miR-1302 MIMAT0005890 UUGGGACAuacuuaugcuaaa 777 hsa-miR-1303 MIMAT0005891 UUUAGAGAcggggucuugcucu 778 hsa-miR-1304 MIMAT0005892 UUUGAGGCuacagugagaugug 779 hsa-miR-1305 MIMAT0005893 UUUUCAACucuaaugggagaga 780 hsa-miR-1243 MIMAT0005894 AACUGGAUcaauuauaggagug 781 hsa-miR-548f MIMAT0005895 AAAAACUGuaauuacuuuu 782 hsa-miR-1244 MIMAT0005896 AAGUAGUUgguuuguaugagaugguu 783 hsa-miR-1245 MIMAT0005897 AAGUGAUCuaaaggccuacau 784 hsa-miR-1246 MIMAT0005898 AAUGGAUUuuuggagcagg 785 hsa-miR-1247 MIMAT0005899 ACCCGUCCcguucguccccgga 786 hsa-miR-1248 MIMAT0005900 ACCUUCUUguauaagcacugugcuaaa 787 hsa-miR-1249 MIMAT0005901 ACGCCCUUcccccccuucuuca 788 hsa-miR-1250 MIMAT0005902 ACGGUGCUggauguggccuuu 789 hsa-miR-1251 MIMAT0005903 ACUCUAGCugccaaaggcgcu 790 hsa-miR-1253 MIMAT0005904 AGAGAAGAagaucagccugca 791 hsa-miR-1254 MIMAT0005905 AGCCUGGAagcuggagccugcagu 792 hsa-miR-1255a MIMAT0005906 AGGAUGAGcaaagaaaguagauu 793 hsa-miR-1256 MIMAT0005907 AGGCAUUGacuucucacuagcu 794 hsa-miR-1257 MIMAT0005908 AGUGAAUGauggguucugacc 795 hsa-miR-1258 MIMAT0005909 AGUUAGGAuuaggucguggaa 796 hsa-miR-1259 MIMAT0005910 AUAUAUGAugacuuagcuuuu 797 hsa-miR-1260 MIMAT0005911 AUCCCACCucugccacca 798 hsa-miR-548g MIMAT0005912 AAAACUGUaauuacuuuuguac 799 hsa-miR-1261 MIMAT0005913 AUGGAUAAggcuuuggcuu 800 hsa-miR-1262 MIMAT0005914 AUGGGUGAauuuguagaaggau 801 hsa-miR-1263 MIMAT0005915 AUGGUACCcuggcauacugagu 802 hsa-miR-548n MIMAT0005916 CAAAAGUAauuguggauuuugu 803 hsa-miR-548m MIMAT0005917 CAAAGGUAuuugugguuuuug 804 hsa-miR-1265 MIMAT0005918 CAGGAUGUggucaaguguuguu 805 hsa-miR-548o MIMAT0005919 CCAAAACUgcaguuacuuuugc 806 hsa-miR-1266 MIMAT0005920 CCUCAGGGcuguagaacagggcu 807 hsa-miR-1267 MIMAT0005921 CCUGUUGAaguguaaucccca 808 hsa-miR-1268 MIMAT0005922 CGGGCGUGgugguggggg 809 hsa-miR-1269 MIMAT0005923 CUGGACUGagccgugcuacugg 810 hsa-miR-1270 MIMAT0005924 CUGGAGAUauggaagagcugugu 811 hsa-miR-1272 MIMAT0005925 GAUGAUGAuggcagcaaauucugaaa 812 hsa-miR-1273 MIMAT0005926 GGGCGACAaagcaagacucuuucuu 813 hsa-miR-1274a MIMAT0005927 GUCCCUGUucaggcgcca 814 hsa-miR-548h MIMAT0005928 AAAAGUAAucgcgguuuuuguc 815 hsa-miR-1275 MIMAT0005929 GUGGGGGAgaggcuguc 816 hsa-miR-1276 MIMAT0005930 UAAAGAGCccuguggagaca 817 hsa-miR-302e MIMAT0005931 UAAGUGCUuccaugcuu 818 hsa-miR-302f MIMAT0005932 UAAUUGCUuccauguuu 819 hsa-miR-1277 MIMAT0005933 UACGUAGAuauauauguauuuu 820 hsa-miR-548p MIMAT0005934 UAGCAAAAacugcaguuacuuu 821 hsa-miR-548i MIMAT0005935 AAAAGUAAuugcggauuuugcc 822 hsa-miR-1278 MIMAT0005936 UAGUACUGugcauaucaucuau 823 hsa-miR-1279 MIMAT0005937 UCAUAUUGcuucuuucu 824 hsa-miR-1274b MIMAT0005938 UCCCUGUUcgggcgcca 825 hsa-miR-1281 MIMAT0005939 UCGCCUCCuccucuccc 826 hsa-miR-1282 MIMAT0005940 UCGUUUGCcuuuuucugcuu 827 hsa-miR-1284 MIMAT0005941 UCUAUACAgacccuggcuuuuc 828 hsa-miR-1288 MIMAT0005942 UGGACUGCccugaucuggaga 829 hsa-miR-1292 MIMAT0005943 UGGGAACGgguuccggcagacgcug 830 hsa-miR-1252 MIMAT0005944 AGAAGGAAauugaauucauuua 831 hsa-miR-1255b MIMAT0005945 CGGAUGAGcaaagaaagugguu 832 hsa-miR-1280 MIMAT0005946 UCCCACCGcugccaccc 833 hsa-miR-1308 MIMAT0005947 GCAUGGGUgguucagugg 834 hsa-miR-664* MIMAT0005948 ACUGGCUAgggaaaaugauuggau 835 hsa-miR-664 MIMAT0005949 UAUUCAUUuauccccagccuaca 836 hsa-miR-1306 MIMAT0005950 ACGUUGGCucugguggug 837 hsa-miR-1307 MIMAT0005951 ACUCGGCGuggcgucggucgug 838 hsa-miR-513b MIMAT0005788 UUCACAAGgaggugucauuuau 839 hsa-miR-513c MIMAT0005789 UUCUCAAGgaggugucguuuau 840 hsa-miR-1321 MIMAT0005952 CAGGGAGGugaaugugau 841 hsa-miR-1322 MIMAT0005953 GAUGAUGCugcugaugcug 842 hsa-miR-720 MIMAT0005954 UCUCGCUGgggccucca 843 hsa-miR-1197 MIMAT0005955 UAGGACACauggucuacuucu 844 hsa-miR-1324 MIMAT0005956 CCAGACAGaauucuaugcacuuuc 845 hsa-miR-1469 MIMAT0007347 CUCGGCGCggggcgcgggcucc 846 hsa-miR-1470 MIMAT0007348 GCCCUCCGcccgugcaccccg 847 hsa-miR-1471 MIMAT0007349 GCCCGCGUguggagccaggugu 848 hsa-miR-1537 MIMAT0007399 AAAACCGUcuaguuacaguugu 849 hsa-miR-1538 MIMAT0007400 CGGCCCGGgcugcugcuguuccu 850 hsa-miR-1539 MIMAT0007401 UCCUGCGCgucccagaugccc 851 hsa-miR-103-as MIMAT0007402 UCAUAGCCcuguacaaugcugcu 852 hsa-miR-320d MIMAT0006764 AAAAGCUGgguugagagga 853 hsa-miR-1825 MIMAT0006765 UCCAGUGCccuccucucc 854 hsa-miR-1826 MIMAT0006766 AUUGAUCAucgacacuucgaacgcaau 855 hsa-miR-1827 MIMAT0006767 UGAGGCAGuagauugaau 856 hsa-miR-1908 MIMAT0007881 CGGCGGGGacggcgauugguc 857 hsa-miR-1909* MIMAT0007882 UGAGUGCCggugccugcccug 858 hsa-miR-1909 MIMAT0007883 CGCAGGGGccgggugcucaccg 859 hsa-miR-1910 MIMAT0007884 CCAGUCCUgugccugccgccu 860 hsa-miR-1911 MIMAT0007885 UGAGUACCgccaugucuguuggg 861 hsa-miR-1911* MIMAT0007886 CACCAGGCauuguggucucc 862 hsa-miR-1912 MIMAT0007887 UACCCAGAgcaugcagugugaa 863 hsa-miR-1913 MIMAT0007888 UCUGCCCCcuccgcugcugcca 864 hsa-miR-1914 MIMAT0007889 CCCUGUGCccggcccacuucug 865 hsa-miR-1914* MIMAT0007890 GGAGGGGUcccgcacugggagg 866 hsa-miR-1915* MIMAT0007891 ACCUUGCCuugcugcccgggcc 867 hsa-miR-1915 MIMAT0007892 CCCCAGGGcgacgcggcggg 868 hsa-miR-1972 MIMAT0009447 UCAGGCCAggcacaguggcuca 869 hsa-miR-1973 MIMAT0009448 ACCGUGCAaagguagcaua 870 hsa-miR-1975 MIMAT0009450 CCCCCACAaccgcgcuugacuagcu 871 hsa-miR-1976 MIMAT0009451 CCUCCUGCccuccuugcugu 872 hsa-miR-1979 MIMAT0009454 CUCCCACUgcuucacuugacua 873 hsa-miR-2052 MIMAT0009977 UGUUUUGAuaacaguaaugu 874 hsa-miR-2053 MIMAT0009978 GUGUUAAUuaaaccucuauuuac 875 hsa-miR-2054 MIMAT0009979 CUGUAAUAuaaauuuaauuuauu 876 hsa-miR-2110 MIMAT0010133 UUGGGGAAacggccgcugagug 877 hsa-miR-2114 MIMAT0011156 UAGUCCCUuccuugaagcgguc 878 hsa-miR-2114* MIMAT0011157 CGAGCCUCaagcaagggacuu 879 hsa-miR-2115 MIMAT0011158 AGCUUCCAugacuccugaugga 880 hsa-miR-2115* MIMAT0011159 CAUCAGAAuucauggaggcuag 881 hsa-miR-2116 MIMAT0011160 GGUUCUUAgcauaggaggucu 882 hsa-miR-2116* MIMAT0011161 CCUCCCAUgccaagaacuccc 883 hsa-miR-2117 MIMAT0011162 UGUUCUCUuugccaaggacag 884 hsa-miR-548q MIMAT0011163 GCUGGUGCaaaaguaauggcgg 885 hsa-miR-2276 MIMAT0011775 UCUGCAAGugucagaggcgagg 886 hsa-miR-2277 MIMAT0011777 UGACAGCGcccugccuggcuc 887 hsa-miR-2278 MIMAT0011778 GAGAGCAGuguguguugccugg 888 hsa-miR-711 MIMAT0012734 GGGACCCAgggagagacguaag 889 hsa-miR-718 MIMAT0012735 CUUCCGCCccgccgggcgucg 890 hsa-miR-2861 MIMAT0013802 GGGGCCUGgcggugggcgg 891 hsa-miR-2909 MIMAT0013863 GUUAGGGCcaacaucucuugg 892 hsa-miR-3115 MIMAT0014977 AUAUGGGUuuacuaguuggu 893 hsa-miR-3116 MIMAT0014978 UGCCUGGAacauaguagggacu 894 hsa-miR-3117 MIMAT0014979 AUAGGACUcauauagugccag 895 hsa-miR-3118 MIMAT0014980 UGUGACUGcauuaugaaaauucu 896 hsa-miR-3119 MIMAT0014981 UGGCUUUUaacuuugauggc 897 hsa-miR-3120 MIMAT0014982 CACAGCAAguguagacaggca 898 hsa-miR-3121 MIMAT0014983 UAAAUAGAguaggcaaaggaca 899 hsa-miR-3122 MIMAT0014984 GUUGGGACaagaggacggucuu 900 hsa-miR-3123 MIMAT0014985 CAGAGAAUuguuuaauc 901 hsa-miR-3124 MIMAT0014986 UUCGCGGGcgaaggcaaaguc 902 hsa-miR-548s MIMAT0014987 AUGGCCAAaacugcaguuauuuu 903 hsa-miR-3125 MIMAT0014988 UAGAGGAAgcuguggagaga 904 hsa-miR-3126-5p MIMAT0014989 UGAGGGACagaugccagaagca 905 hsa-miR-3126-3p MIMAT0015377 CAUCUGGCauccgucacacaga 906 hsa-miR-3127 MIMAT0014990 AUCAGGGCuuguggaaugggaag 907 hsa-miR-3128 MIMAT0014991 UCUGGCAAguaaaaaacucucau 908 hsa-miR-3129 MIMAT0014992 GCAGUAGUguagagauugguuu 909 hsa-miR-3130-5p MIMAT0014995 UACCCAGUcuccggugcagcc 910 hsa-miR-3130-3p MIMAT0014994 GCUGCACCggagacuggguaa 911 hsa-miR-3131 MIMAT0014996 UCGAGGACugguggaagggccuu 912 hsa-miR-3132 MIMAT0014997 UGGGUAGAgaaggagcucagagga 913 hsa-miR-3133 MIMAT0014998 UAAAGAACucuuaaaacccaau 914 hsa-miR-378b MIMAT0014999 ACUGGACUuggaggcagaa 915 hsa-miR-3134 MIMAT0015000 UGAUGGAUaaaagacuacauauu 916 hsa-miR-3135 MIMAT0015001 UGCCUAGGcugagacugcagug 917 hsa-miR-466 MIMAT0015002 AUACACAUacacgcaacacacau 918 hsa-miR-3136 MIMAT0015003 CUGACUGAauagguagggucauu 919 hsa-miR-544b MIMAT0015004 ACCUGAGGuugugcauuucuaa 920 hsa-miR-3137 MIMAT0015005 UCUGUAGCcugggagcaauggggu 921 hsa-miR-3138 MIMAT0015006 UGUGGACAgugagguagagggagu 922 hsa-miR-3139 MIMAT0015007 UAGGAGCUcaacagaugccuguu 923 hsa-miR-3140 MIMAT0015008 AGCUUUUGggaauucagguagu 924 hsa-miR-548t MIMAT0015009 CAAAAGUGaucgugguuuuug 925 hsa-miR-3141 MIMAT0015010 GAGGGCGGguggaggagga 926 hsa-miR-3142 MIMAT0015011 AAGGCCUUucugaaccuucaga 927 hsa-miR-3143 MIMAT0015012 AUAACAUUguaaagcgcuucuuucg 928 hsa-miR-548u MIMAT0015013 CAAAGACUgcaauuacuuuugcg 929 hsa-miR-3144-5p MIMAT0015014 AGGGGACCaaagagauauauag 930 hsa-miR-3144-3p MIMAT0015015 AUAUACCUguucggucucuuua 931 hsa-miR-3145 MIMAT0015016 AGAUAUUUugaguguuuggaauug 932 hsa-miR-1273c MIMAT0015017 GGCGACAAaacgagacccuguc 933 hsa-miR-3146 MIMAT0015018 CAUGCUAGgauagaaagaaugg 934 hsa-miR-3147 MIMAT0015019 GGUUGGGCagugaggaggguguga 935 hsa-miR-548v MIMAT0015020 AGCUACAGuuacuuuugcacca 936 hsa-miR-3148 MIMAT0015021 UGGAAAAAacuggugugugcuu 937 hsa-miR-3149 MIMAT0015022 UUUGUAUGgauauguguguguau 938 hsa-miR-3150 MIMAT0015023 CUGGGGAGauccucgagguugg 939 hsa-miR-3151 MIMAT0015024 GGUGGGGCaaugggaucaggu 940 hsa-miR-3152 MIMAT0015025 UGUGUUAGaauaggggcaauaa 941 hsa-miR-3153 MIMAT0015026 GGGGAAAGcgaguagggacauuu 942 hsa-miR-3074 MIMAT0015027 GAUAUCAGcucaguaggcaccg 943 hsa-miR-3154 MIMAT0015028 CAGAAGGGgaguugggagcaga 944 hsa-miR-3155 MIMAT0015029 CCAGGCUCugcagugggaacu 945 hsa-miR-3156 MIMAT0015030 AAAGAUCUggaagugggagaca 946 hsa-miR-3157 MIMAT0015031 UUCAGCCAggcuagugcagucu 947 hsa-miR-3158 MIMAT0015032 AAGGGCUUccucucugcaggac 948 hsa-miR-3159 MIMAT0015033 UAGGAUUAcaagugucggccac 949 hsa-miR-3160 MIMAT0015034 AGAGCUGAgacuagaaagccca 950 hsa-miR-3161 MIMAT0015035 CUGAUAAGaacagaggcccagau 951 hsa-miR-3162 MIMAT0015036 UUAGGGAGuagaaggguggggag 952 hsa-miR-3163 MIMAT0015037 UAUAAAAUgagggcaguaagac 953 hsa-miR-3164 MIMAT0015038 UGUGACUUuaagggaaauggcg 954 hsa-miR-3165 MIMAT0015039 AGGUGGAUgcaaugugaccuca 955 hsa-miR-3166 MIMAT0015040 CGCAGACAaugccuacuggccua 956 hsa-miR-1260b MIMAT0015041 AUCCCACCacugccaccau 957 hsa-miR-3167 MIMAT0015042 AGGAUUUCagaaauacuggugu 958 hsa-miR-3168 MIMAT0015043G AGUUCUAcagucagac 959 hsa-miR-3169 MIMAT0015044 UAGGACUGugcuuggcacauag 960 hsa-miR-3170 MIMAT0015045 CUGGGGUUcugagacagacagu 961 hsa-miR-3171 MIMAT0015046 AGAUGUAUggaaucuguauauauc 962 hsa-miR-3172 MIMAT0015047 UGGGGUUUugcaguccuua 963 hsa-miR-3173 MIMAT0015048 AAAGGAGGaaauaggcaggcca 964 hsa-miR-1193 MIMAT0015049 GGGAUGGUagaccggugacgugc 965 hsa-miR-323b-5p MIMAT0001630 AGGUUGUCcguggugaguucgca 966 hsa-miR-323b-3p MIMAT0015050 CCCAAUACacggucgaccucuu 967 hsa-miR-3174 MIMAT0015051 UAGUGAGUuagagaugcagagcc 968 hsa-miR-3175 MIMAT0015052 CGGGGAGAgaacgcagugacgu 969 hsa-miR-3176 MIMAT0015053 ACUGGCCUgggacuaccgg 970 hsa-miR-3177 MIMAT0015054 UGCACGGCacuggggacacgu 971 hsa-miR-3178 MIMAT0015055 GGGGCGCGgccggaucg 972 hsa-miR-3179 MIMAT0015056 AGAAGGGGugaaauuuaaacgu 973 hsa-miR-3180-5p MIMAT0015057 CUUCCAGAcgcuccgccccacgucg 974 hsa-miR-3180-3p MIMAT0015058 UGGGGCGGagcuuccggaggcc 975 hsa-miR-548w MIMAT0015060 AAAAGUAAcugcgguuuuugccu 976 hsa-miR-3181 MIMAT0015061 AUCGGGCCcucggcgccgg 977 hsa-miR-3182 MIMAT0015062 GCUUCUGUaguguaguc 978 hsa-miR-3183 MIMAT0015063 GCCUCUCUcggagucgcucgga 979 hsa-miR-3184 MIMAT0015064 UGAGGGGCcucagaccgagcuuuu 980 hsa-miR-3185 MIMAT0015065 AGAAGAAGgcggucggucugcgg 981 hsa-miR-3065-5p MIMAT0015066 UCAACAAAaucacugaugcugga 982 hsa-miR-3065-3p MIMAT0015378 UCAGCACCaggauauuguuggag 983 hsa-miR-3186-5p MIMAT0015067 CAGGCGUCugucuacguggcuu 984 hsa-miR-3186-3p MIMAT0015068 UCACGCGGagagauggcuuug 985 hsa-miR-3187 MIMAT0015069 UUGGCCAUggggcugcgcgg 986 hsa-miR-3188 MIMAT0015070 AGAGGCUUugugcggauacgggg 987 hsa-miR-3189 MIMAT0015071 CCCUUGGGucugaugggguag 988 hsa-miR-320e MIMAT0015072 AAAGCUGGguugagaagg 989 hsa-miR-3190-5p MIMAT0015073 UGUGGAAGguagacggccagaga 990 hsa-miR-3190-3p MIMAT0015074 UGGAAGGUagacggccagagag 991 hsa-miR-3191 MIMAT0015075 UGGGGACGuagcuggccagacag 992 hsa-miR-3192 MIMAT0015076 UCUGGGAGguuguagcaguggaa 993 hsa-miR-3193 MIMAT0015077 UCCUGCGUaggaucugaggagu 994 hsa-miR-3194 MIMAT0015078 GGCCAGCCaccaggagggcug 995 hsa-miR-3195 MIMAT0015079 CGCGCCGGgcccggguu 996 hsa-miR-3196 MIMAT0015080 CGGGGCGGcaggggccuc 997 hsa-miR-548x MIMAT0015081 UAAAAACUgcaauuacuuuca 998 hsa-miR-3197 MIMAT0015082 GGAGGCGCaggcucggaaaggcg 999 hsa-miR-3198 MIMAT0015083 GUGGAGUCcuggggaauggaga 1000 hsa-miR-3199 MIMAT0015084 AGGGACUGccuuaggagaaaguu 1001 hsa-miR-3200 MIMAT0015085 CACCUUGCgcuacucaggucug 1002 hsa-miR-3201 MIMAT0015086 GGGAUAUGaagaaaaau 1003 hsa-miR-514b-5p MIMAT0015087 UUCUCAAGagggaggcaaucau 1004 hsa-miR-514b-3p MIMAT0015088 AUUGACACcucugugagugga 1005 hsa-miR-3202 MIMAT0015089 UGGAAGGGagaagagcuuuaau 1006 hsa-miR-1273d MIMAT0015090 GAACCCAUgagguugaggcugcagu 1007 hsa-miR-4295 MIMAT0016844 CAGUGCAAuguuuuccuu 1008 hsa-miR-4296 MIMAT0016845 AUGUGGGCucaggcuca 1009 hsa-miR-4297 MIMAT0016846 UGCCUUCCugucugug 1010 hsa-miR-378c MIMAT0016847 ACUGGACUuggagucagaagagugg 1011 hsa-miR-4293 MIMAT0016848 CAGCCUGAcaggaacag 1012 hsa-miR-4294 MIMAT0016849 GGGAGUCUacagcaggg 1013 hsa-miR-4301 MIMAT0016850 UCCCACUAcuucacuuguga 1014 hsa-miR-4299 MIMAT0016851 GCUGGUGAcaugagaggc 1015 hsa-miR-4298 MIMAT0016852 CUGGGACAggaggaggaggcag 1016 hsa-miR-4300 MIMAT0016853 UGGGAGCUggacuacuuc 1017 hsa-miR-4304 MIMAT0016854 CCGGCAUGuccagggca 1018 hsa-miR-4302 MIMAT0016855 CCAGUGUGgcucagcgag 1019 hsa-miR-4303 MIMAT0016856 UUCUGAGCugaggacag 1020 hsa-miR-4305 MIMAT0016857 CCUAGACAccuccaguuc 1021 hsa-miR-4306 MIMAT0016858 UGGAGAGAaaggcagua 1022 hsa-miR-4309 MIMAT0016859 CUGGAGUCuaggauucca 1023 hsa-miR-4307 MIMAT0016860 AAUGUUUUuuccuguuucc 1024 hsa-miR-4308 MIMAT0016861 UCCCUGGAguuucuucuu 1025 hsa-miR-4310 MIMAT0016862 GCAGCAUUcauguccc 1026 hsa-miR-4311 MIMAT0016863 GAAAGAGAgcugagugug 1027 hsa-miR-4312 MIMAT0016864 GGCCUUGUuccugucccca 1028 hsa-miR-4313 MIMAT0016865 AGCCCCCUggccccaaaccc 1029 hsa-miR-4315 MIMAT0016866 CCGCUUUCugagcuggac 1030 hsa-miR-4316 MIMAT0016867 GGUGAGGCuagcuggug 1031 hsa-miR-4314 MIMAT0016868 CUCUGGGAaaugggacag 1032 hsa-miR-4318 MIMAT0016869 CACUGUGGguacaugcu 1033 hsa-miR-4319 MIMAT0016870 UCCCUGAGcaaagccac 1034 hsa-miR-4320 MIMAT0016871 GGGAUUCUguagcuuccu 1035 hsa-miR-4317 MIMAT0016872 ACAUUGCCagggaguuu 1036 hsa-miR-4322 MIMAT0016873 CUGUGGGCucagcgcgugggg 1037 hsa-miR-4321 MIMAT0016874 UUAGCGGUggaccgcccugcg 1038 hsa-miR-4323 MIMAT0016875 CAGCCCCAcagccucaga 1039 hsa-miR-4324 MIMAT0016876 CCCUGAGAcccuaaccuuaa 1040 hsa-miR-4256 MIMAT0016877 AUCUGACCugaugaaggu 1041 hsa-miR-4257 MIMAT0016878 CCAGAGGUggggacugag 1042 hsa-miR-4258 MIMAT0016879 CCCCGCCAccgccuugg 1043 hsa-miR-4259 MIMAT0016880 CAGUUGGGucuaggggucagga 1044 hsa-miR-4260 MIMAT0016881 CUUGGGGCauggaguccca 1045 hsa-miR-4253 MIMAT0016882 AGGGCAUGuccagggggu 1046 hsa-miR-4251 MIMAT0016883 CCUGAGAAaagggccaa 1047 hsa-miR-4254 MIMAT0016884 GCCUGGAGcuacuccaccaucuc 1048 hsa-miR-4255 MIMAT0016885 CAGUGUUCagagaugga 1049 hsa-miR-4252 MIMAT0016886 GGCCACUGagucagcacca 1050 hsa-miR-4325 MIMAT0016887 UUGCACUUgucucaguga 1051 hsa-miR-4326 MIMAT0016888 UGUUCCUCugucucccagac 1052 hsa-miR-4327 MIMAT0016889 GGCUUGCAugggggacugg 1053 hsa-miR-4261 MIMAT0016890 AGGAAACAgggaccca 1054 hsa-miR-4265 MIMAT0016891 CUGUGGGCucagcucuggg 1055 hsa-miR-4266 MIMAT0016892 CUAGGAGGccuuggcc 1056 hsa-miR-4267 MIMAT0016893 UCCAGCUCgguggcac 1057 hsa-miR-4262 MIMAT0016894 GACAUUCAgacuaccug 1058 hsa-miR-2355 MIMAT0016895 AUCCCCAGauacaauggacaa 1059 hsa-miR-4268 MIMAT0016896 GGCUCCUCcucucaggaugug 1060 hsa-miR-4269 MIMAT0016897 GCAGGCACagacagcccuggc 1061 hsa-miR-4263 MIMAT0016898 AUUCUAAGugccuuggcc 1062 hsa-miR-4264 MIMAT0016899 ACUCAGUCauggucauu 1063 hsa-miR-4270 MIMAT0016900 UCAGGGAGucaggggagggc 1064 hsa-miR-4271 MIMAT0016901 GGGGGAAGaaaaggugggg 1065 hsa-miR-4272 MIMAT0016902 CAUUCAACuagugauugu 1066 hsa-miR-4273 MIMAT0016903 GUGUUCUCugauggacag 1067 hsa-miR-4276 MIMAT0016904 CUCAGUGAcucaugugc 1068 hsa-miR-4275 MIMAT0016905 CCAAUUACcacuucuuu 1069 hsa-miR-4274 MIMAT0016906 CAGCAGUCccucccccug 1070 hsa-miR-4281 MIMAT0016907 GGGUCCCGgggagggggg 1071 hsa-miR-4277 MIMAT0016908 GCAGUUCUgagcacaguacac 1072 hsa-miR-4279 MIMAT0016909 CUCUCCUCccggcuuc 1073 hsa-miR-4278 MIMAT0016910 CUAGGGGGuuugcccuug 1074 hsa-miR-4280 MIMAT0016911 GAGUGUAGuucugagcagagc 1075 hsa-miR-4282 MIMAT0016912 UAAAAUUUgcauccagga 1076 hsa-miR-4285 MIMAT0016913 GCGGCGAGuccgacucau 1077 hsa-miR-4283 MIMAT0016914 UGGGGCUCagcgaguuu 1078 hsa-miR-4284 MIMAT0016915 GGGCUCACaucaccccau 1079 hsa-miR-4286 MIMAT0016916 ACCCCACUccugguacc 1080 hsa-miR-4287 MIMAT0016917 UCUCCCUUgagggcacuuu 1081 hsa-miR-4288 MIMAT0016918 UUGUCUGCugaguuucc 1082 hsa-miR-4292 MIMAT0016919 CCCCUGGGccggccuugg 1083 hsa-miR-4289 MIMAT0016920 GCAUUGUGcagggcuauca 1084 hsa-miR-4290 MIMAT0016921 UGCCCUCCuuucuucccuc 1085 hsa-miR-4291 MIMAT0016922 UUCAGCAGgaacagcu 1086 hsa-miR-4329 MIMAT0016923 CCUGAGACccuaguuccac 1087 hsa-miR-4330 MIMAT0016924 CCUCAGAUcagagccuugc 1088 hsa-miR-500b MIMAT0016925 AAUCCUUGcuaccugggu 1089 hsa-miR-4328 MIMAT0016926 CCAGUUUUcccaggauu 1090

EXAMPLES Example 1

Segmented miRNA Mimetics

MicroRNAs (miRNAs) are a class of ˜22 nt noncoding RNAs that play important roles in regulating gene expression in plants and animals. MicroRNAs are usually produced by a process in which a RNA pol II transcript is cut by Drosha to produce a precursor hairpin, which is cut by Dicer in the cytoplasm to produce a two stranded duplex that is incorporated into Argonaute (Ago) proteins. After elimination of the passenger strand by cleavage or helicase activity, the guide strand can then bind to complementary target RNAs. Studies have found that the most prevalent aspect of miRNA target recognition is complementary binding to a target 3′ UTR by the miRNA seed region (positions 2 through 8 at the 5′ end of the guide strand), leading to downregulation at mRNA and protein levels.

Ago2 mediated cleavage of the passenger strand has been found to be important for assembly of siRNAs and some miRNAs and a nicked passenger strand was found to rescue the activity of an siRNA containing a phosphorothioate bond that prevented passenger strand cleavage by Ago. This concept has been applied to the design of siRNAs (Bramsen et al., 2007 supra), where passenger segmentation was found to maintain siRNA activity (while eliminating passenger strand activity), while guide segmentation was found to eliminate the desired siRNA activity.

Applicant has surprisingly found that, as opposed to siRNA, segmentation is well tolerated in the guide strand of microRNA. Applicant demonstrates herein that this segmentation provides for the alternative design of various miRNA mimetics that include nicks and gaps, as well as substitutions and insertions that can confer additional properties toward therapeutic use.

Materials and Methods

Oligonucleotides used to obtain data in this Example were synthesized at Sigma-Aldrich or Merck & Co. using standard methodologies. Annealing was accomplished by mixing single stranded RNA at 10 uM in 10 mM TrisHCl/50 mM NaCl and heating at 95° C. for 2 minutes before slowly cooling to 37° C. over the course of 1 hour.

The RNA oligonucleotides synthesized are shown in the following Table II.

TABLE II  Name Sequence SEQ ID NO. G UUAAGGCACGCGGUGAAUGCCA 1091 P GCAUUCACCGCGUGCCUUAAAU 1092 G10 UUAAGGCACG 1093 G.10 CGGUGAAUGCCA 1094 G.9 GGUGAAUGCCA 1095 P10 GCAUUCACCG 1096 P.10 CGUGCCUUAAAU 1097 G10i UUAAGGCACG-iB 1098 G10Cy3 UUAAGGCACG-Cy3 1099 58-mer GCGUUCACCGCGGACCUUGAUUUAAAUGU 1100 CCAUACAAUUAAGGCACGCGGUGAAUGCC 48-mer GCGUUCACCGCGGACCUUGAUUUAAAUGU 1101 CCAUACAAUUAAGGCACGCGGUGAAUGCC 10-mer GGUGAAUGCC 1102

Sequences in the table above are shown in 5′ to 3′ orientation. “iB” denotes an inverted abasic, while “Cy3” denotes a Cy3 fluorescent dye molecule.

HCT-116 cells were cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, plated in 96-well culture plates at a density of 25,000 cells/well 24 hours prior to transfection, and then transfected using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine 2000 (Invitrogen) with a final concentration of our miRNAs ranging from 30 nM down to 0.01 nM along a 12-point titration curve. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CT™ Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using CD164-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector. Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization.

miRNAs were co-transfected with siCHECK2 vectors (Genscript) containing cloned target inserts, as shown in FIG. 14, consisting of a tandem repeat of a seed match to miR-124 (2×7a), a seed match containing additional 3′ complementarity to positions 13-17 of miR-124 (2×7a3p), or a full-length match to miR-124 (2×FL). A seed match with a two-base mutation (2×7aMutant) was used as a control. Twenty-four hours after transfection, transfection medium was replaced with fresh growth medium. Forty-eight hours after transfection, cells were lysed and both firefly and Renilla luciferase activity were measured using the Dual-Glo™ Luciferase Assay System (Promega) on a Wallac EnVision 2103 Multilabel Reader (PerkinElmer).

HCT-116 cells were transfected with 10 nM miRNA duplex as described previously (Jackson et al 2006). RNA was extracted using RNeasy (Qiagen), amplified using the Ovation protocol (Nugen), and profiled on custom Affymetrix arrays (Rosetta Custom Human 1.0, Affymetrix). Array signals were analyzed with Affymetrix GeneChip Operating Software and Affymetrix Power Tools. UTR hexamer analysis was carried out as described previously (Jackson et al 2006).

Results

Structural variants of a miR-124 duplex were tested, wherein a nick was introduced 10 nucleotides from the 5′ end of either the guide (or miRNA) or passenger (or miRNA*) strand (FIG. 12A). Duplexes were transfected into cells and changes in the mRNA levels of miR-124 target CD164 were measured. Division of the passenger strand (G/P10.10) had little effect on miR-124 activity (FIG. 12B), leading to a slight increase in EC50 (0.12 nM to 0.29 nM). Division of the guide strand (G10.10/P) still allowed for miR-124 activity (EC50 of 0.22 nM), indicating that a continuous guide strand is not required for miRNA RISC activity. The addition of a one base gap between the guide halves, or capping of the junction with a Cy3 dye or an inverted abasic residue, still gave miR-124 activity, indicating that this activity was not a result of ligation of the guide halves. Toleration of guide strand segmentation is not a property only of the miR-124 sequence, as division of the guide strand in a miR-34 duplex still allowed for miR-34a activity (FIG. 9).

Microarrays were used to profile cells transfected with a miR-124 duplex containing the divided guide strand (FIG. 13A) to further confirm the targeting activity of a segmented microRNA duplex, in this instance at a genome-wide level. Analysis of the 3′ UTR sequences of the downregulated genes shows that the most significantly enriched hexamer is GCCTTA, which corresponds to the seed sequence of miR-124. Profiling of the effects of the segmented miR-124 duplex G10.10/P showed correlation (r=0.90) with profiling of the effects of the fully intact duplex (G/P), consistent with the preservation of the bulk of miR-124 targeting (FIG. 13B).

Previous work analyzing microarray profiles has shown that although the preponderance of miRNA targeting is due to seed sequence activity, a much smaller degree of downregulation can be attributed to other contributing factors, among them the supplementary binding of positions 13-16 of the miRNA. Microarray profiling of miR-124 targets containing supplementary 3′ binding was analyzed, and a shift following guide strand segmentation was detected that was indicative of a loss of supplementary 3′ binding activity in the divided miRNA (FIG. 16).

The effects of segmented miRNAs on luciferase reporter vectors were tested, using miRNAs whose 3′ UTRs had been engineered (FIG. 14A) to contain miRNA-complementary sites that constituted a full-length match (2×FL), a seed sequence match (2×7a), or a seed sequence plus supplementary 3′ match (2×7a3p). For an intact duplex (G/P), the repressive activity of miR-124 on luciferase activity was highest for 2×FL and followed the order 2×FL-2×7a3p>2×7a. (FIG. 14B) Similar behavior was seen with a segmented passenger strand (G/P10.10, FIG. 14C). However, when the guide strand was divided (G10.10/P, FIG. 14D), the activities of the 2×FL and 2×7a3p reporters became equivalent to that of 2×7a, showing that the discontinuity at position 10 of the guide strand prevents productive 3′ binding, while permitting seed-based activity from the 5′ half.

Activity of a segmented guide strand was tested in the context of a hairpin sequence that was designed to emulate the natural miR-124 hairpin. Appreciable activity was observed following guide strand division (FIG. 15B), indicating that processing of a hairpin into an Ago-recognizable duplex can occur in spite of a break in the guide strand.

Example 2

Segmented miRNA Mimetics Targeted to CD164

MicroRNAs can down-regulate gene expression by inhibiting translation of their target transcripts and/or mediating the degradation of these transcripts. This Example demonstrates that certain of the segmented miRNA mimetic constructs according to the present disclosure designed based on the corresponding naturally-occurring miRNAs are capable of doing the same. This example also indicates that segmented miRNA mimetics can comprise one or more locked nucleic acids (named “(L)”, underlined nucleotides are locked nucleic acid residues in Table III, IV, V and VI). Nicks are marked within the sequence as “(nick).” Gaps are marked within the sequence as “(

)” with each box indicating a one nucleotide gap.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table III, passenger strand shown on top and guide strand on bottom) and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICER^(ex5), a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target gene, CD164, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96).

Passenger strand sequences in Table III are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE III  SEQ ID NOs. Name Sequence P G miR- (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 124/124(p)/124 ACCGUAAGUGGCGCACGGAAUU (guide) (g) (124p)10(L).10 (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1107/ 1091 (L)/(124g) ACCGUAAGUGGCGCACGGAAUU (guide) 1108 (124p)10.10/ (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1096/ 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) 1097 (124p)12(L).8(L)/ (passenger) GCAUUCACCGCG(nick)UGCCUUAAAU  1105/ 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) 1106 (124p)10(L).8(L)/ (passenger) GCAUUCACCG(

)UGCCUUAAAU  1107/ 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) 1109 (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1103/ (L).10(L) ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1104 (124p)/(124g)10.10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1093/ ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1094 (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1103/ (L).9(L) ACCGUAAGUGG(

)GCACGGAAUU (guide) 1110 (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1103/ (L).8(L) ACCGUAAGUG(

)GCACGGAAUU (guide) 1112 (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1103 (L).7(L) ACCGUAAGU(

)GCACGGAAUU (guide) (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1093 (L).7 ACCGUAAGU(

)GCACGGAAUU (guide) (124p)/(124g)11 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1111/ (L).9(L) ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1110 (124p)/(124g)11 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1111/ (L).8(L) ACCGUAAGUG(

)CGCACGGAAUU (guide) 1112 (124p)/(124g)11 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1111 (L).7(L) ACCGUAAGU(

)CGCACGGAAUU (guide) (124p)/(124g)12 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1113/ (L).8(L) ACCGUAAGUG(nick)GCGCACGGAAUU (guide) 1112 (124p)/(124g)12 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1113 (L).7(L) ACCGUAAGU(

)GCGCACGGAAUU (guide) (124p)/(124g)13 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1114 (L).7(L) ACCGUAAGU(nick)GGCGCACGGAAUU (guide)

Results of the activities are shown in FIG. 7. In brief, most of the constructs tested demonstrated a capacity of knocking down CD164, a known target to the naturally-occurring endogenous miR-124. Constructs comprising nicks in one or both strands of the segmented miRNA mimetic demonstrated at least 25% of knockdown (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) achieved by the non-segmented miR-24 positive control.

Example 3

Segmented miRNA Mimetics Targeted to VAMP3

Segmented miRNA mimetics can be designed to include a discontinuity comprising a nick or gap in one or both strands of any miRNA sequence of the invention in which target specific silencing activity is maintained. In the following example, nicks and gaps were introduced into miR-124 miRNA mimetics and downstream target silencing was confirmed.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table IV) and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICERex5, a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target gene, VAMP3, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96).

Passenger strand sequences in Table IV are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE IV  SEQ ID NO(s). Name Sequence P G miR-124/ (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 (124(p)/124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)10(L).10 (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1107/ 1091 (L)/(124g) ACCGUAAGUGGCGCACGGAAUU (guide) 1108 (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1103/ (L).10(L) ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1104 (124p)/(124g)11 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1111/ (L).9(L) ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1110 (124p)10(L).10 (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1107/  1103/ (L)/(124g)10 ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1108 1104 (L).10(L) (124p)10(L).10 (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1107/  1093/ (L)/(124g)10.10 ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1108 1094 (124p)10.10/ (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1096/  1103/ (124g)10(L).10 ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1097 1104 (L) (124p)10.10/ (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1096/  1093/ (124g)10.10 ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1097 1094 (124p)10(L).10 (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1107/  1111/ (L)/(124g)11 ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1108 1110 (L).9(L) (124p)10.10/ (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1096/  1111/ (124g)11(L).9(L) ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1097 1110 (124p)10(L).10 (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1107/  1113/ (L)/(124g)12 ACCGUAAGUG(nick)GCGCACGGAAUU (guide) 1108 1112 (L).8(L) (124p)10.10/ (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1096/  1113/ (124g)12(L).8(L) ACCGUAAGUG(nick)GCGCACGGAAUU (guide) 1097 1112 (124p)10(L).10 (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1107/ 1114 (L)/(124g)13 ACCGUAAGU(nick)GGCGCACGGAAUU (guide) 1108 (L).7(L) (124p)10.10/ (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1096/ 1114 (124g)13(L).7(L) ACCGUAAGU(nick)GGCGCACGGAAUU (guide) 1097 (124p)12(L).8 (passenger) GCAUUCACCGCG(nick)UGCCUUAAAU  1105/  1103/ (L)/(124g)10 ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1106 1104 (L).10(L) (124p)12(L).8 (passenger) GCAUUCACCGCG(nick)UGCCUUAAAU  1105/  1103/ (L)/(124g)10.10 ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1106 1104 (124p)12(L).8 (passenger) GCAUUCACCGCG(nick)UGCCUUAAAU  1105/  1111/ (L)/(124g)11 ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1106 1110 (L).9(L) (124p)12(L).8 (passenger) GCAUUCACCGCG(nick)UGCCUUAAAU  1105/  1113/ (L)/(124g)12 ACCGUAAGUG(nick)GCGCACGGAAUU (guide) 1106 1112 (L).8(L) (124p)12(L).8  (passenger) GCAUUCACCGCG(nick)UGCCUUAAAU  1105/ 1114 (L)/(124g)13 ACCGUAAGU(nick)GGCGCACGGAAUU (guide) 1106 (L).7(L)

Results of this example are shown in FIG. 8. A number of the segmented miRNA mimetic constructs of this example, each strand comprising two distinct contiguous stretches of nucleotides, achieved at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the knockdown effect as compared to the non-segmented duplex miR-124 mimetic.

Example 4

Segmented miRNA Mimetics of miR-124 Versus miR-34

Segmented miRNA mimetics can be designed to include a discontinuity comprising a nick or gap in any miRNA sequence of the invention in which the target specificity is maintained. In the following example, nicks and gaps were introduced into miR-124 and miR-34 miRNA mimetics and downstream target specificity determined.

Synthetic duplex mimetic of miR-124 and segmented miR-124 constructs (sequences shown in Table V) and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICER^(ex5), a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target gene, CD 164, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96). The knockdown effect achieved by segmented miRNA-124 was also compared with the knockdown, or the lack thereof, achieved by segmented miRNA-34 and a duplex miR-34 mimetic, which are known to not target CD 164.

Synthetic duplex mimetic of miR-34 and segmented miR-34 constructs (sequences shown in Table V) and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICER^(ex5), a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target gene, TK1, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96). The knockdown effect achieved by segmented miRNA-34 was also compared with the knockdown, or the lack thereof, achieved by segmented miRNA-124 and a duplex miR-124 mimetic, which are known to not target TK1.

The nucleotides that were changed to effectuate the mismatches are indicated in lower case letters in the sequences. Passenger strand sequences in Table V are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE V  SEQ ID NO(s). Name Sequence P G miR-124/(124p)/ (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) miR-124 (blunt (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1115 end) CGUAAGUGGCGCACGGAAUU (guide) miR-124 (shorter (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1116 guide) UAAGUGGCGCACGGAAUU (guide) (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1103/ (L).10(L) ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1104 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1093/ 10.10 ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1094 (124p)10.10/ (passenger) GCAUUCACCG(nick)CGUGCCUUAAAU  1096/ 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) 1097 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1093/ 10.10 ACCGUAAGUGGC(nick)GCACGGAAUU (guide) 1094 (124p)/(124g)11.9 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1117/ ACCGUAAGUGG(nick)CGCACGGAAUU (guide) 1118 (124p)/(124g) (passenger) GCAUcuACCG(nick)CGUGCCUUAAAU  1119/  1093/ 10.10m (mismatch) ACCGUAgaUGGC(nick)GCACGGAAUU (guide) 1097 1120 (124p)/(124g)10 (passenger) GCAUUCACCG(nick)CGUcgCUUAAAU  1096/  1122/ m3.10 (mismatch) ACCGUAAGUGGC(nick)GCAgcGAAUU (guide) 1121 1094 (124p)/(124g)10 (passenger) GCAUUCACCG(nick)CGUGCCUauAAU  1096/  1124/ m5.10 (mismatch) ACCGUAAGUGG(nick)CGCACGGAuaU (guide) 1123 1118 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1117/ 10.9 ACCGUAAGUG(

)CGCACGGAAUU (guide) 1125 (124p)/(124g)10 (passenger) GCAUUCACCGCGUcgCUUAAAU 1126 1127 m3.9 (mismatch) ACCGUAAGU(

)GCGCAgcGAAUU (guide) (124p)/(124g)10 (passenger) GCAUUCACCG(nick)CGUGCCUauAAU  1096/  1128/ m5.9 (mismatch) ACCGUAAGUG(nick)GCGCACGGAuaU (guide) 1112 miR- (passenger) CACGAGCUAAGACACUGCUAAU 1093 1094 34a/(34p)/(34g) UGGUGCUCGAUUCUGUGACGGU (guide) (34p)/(34g)10.10 (passenger) CACGAGCUAAGACACUGCUAAU 1093  1129/ UGGUGCUCGAUU(nick)CUGUGACGGU (guide) 1130 (34p)/(34g)11.9 (passenger) CACGAGCUAAGACACUGCUAAU 1093  1131/ UGGUGCUCGAU(nick)UCUGUGACGGU (guide) 1132 (34p)/(34g)10.9 (passenger) CACGAGCUAAGACACUGCUAAU 1093  1129/ UGGUGCUCGAU(

)CUGUGACGGU (guide) 1132

The results are presented in FIGS. 9A and 9B. Certain segmented miRNA mimetic constructs of this example, achieved at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the knockdown effect as compared to their respective non-segmented duplex mimetics.

Example 5

Segmented miRNA Mimetics Comprising Abasic Insertions

Segmented miRNA mimetics can be designed to include a discontinuity comprising a nick or gap, in which one or more non-nucleotide moieties of the invention are inserted into the terminal portions of sequence adjacent to the nick or gap. In the following example, abasic moieties were used to cap the internal ends of nucleotide positions in the guide strand of a segmented miRNA mimetic. Likewise, insertions with other non-nucleotide moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table VI), comprising one or more inverted abasic modifications at the internal ends, and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICER^(ex5), a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target genes, CD164 and VAMP3, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96).

The position of the inverted abasic group is indicated as “(i)” in both the names and the sequences of the following table. Passenger strand sequences in Table VI are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE VI SEQ ID NO(s). Name Sequence P G miR-124 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 ACCGUAAGUGGCGCACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1133 7(i).13(i) ACCGUAAGUGGCGCA(i)(nick)(i)CGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1134 8(i).12(i) ACCGUAAGUGGCGC(i)(nick)(i)ACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1135 9(i).11(i) ACCGUAAGUGGCG(i)(nick)(i)CACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1136/ 10(i).10(i) ACCGUAAGUGGC(i)(nick)(i)GCACGGAAUU (guide) 1137 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1138/ 11(i).9(i) ACCGUAAGUGG(i)(nick)(i)CGCACGGAAUU (guide) 1139 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1140/ 12(i).8(i) ACCGUAAGUG(i)(nick)(i)GCGCACGGAAUU (guide) 1141 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1142 13(i).7(i) ACCGUAAGU(i)(nick)(i)GGCGCACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1143 7(i).13 ACCGUAAGUGGCGCA(nick)(i)CGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1144 8(i).12 ACCGUAAGUGGCGC(nick)(i)ACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1145 9(i).11 ACCGUAAGUGGCG(nick)(i)CACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1136/ 10(i).10 ACCGUAAGUGGC(nick)(i)GCACGGAAUU (guide) 1094 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092   1138/ 11(i).9 ACCGUAAGUGG(nick)(i)CGCACGGAAUU (guide) 1118 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1140/ 12(i).8 ACCGUAAGUG(nick)(i)GCGCACGGAAUU (guide) 1125 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1142 13(i).7 ACCGUAAGU(nick)(i)GGCGCACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1133 7.13(i) ACCGUAAGUGGCGCA(i)(nick)CGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1134 8.12(i) ACCGUAAGUGGCGC(i)(nick)ACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1135 9.11(i) ACCGUAAGUGGCG(i)(nick)CACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1093/ 10.10(i) ACCGUAAGUGGC(i)(nick)GCACGGAAUU (guide) 1137 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1117/ 11.9(i) ACCGUAAGUGG(i)(nick)CGCACGGAAUU (guide) 1139 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092  1146/ 12.8(i) ACCGUAAGUG(i)(nick)GCGCACGGAAUU (guide) 1141 (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1147 13.7(i) ACCGUAAGU(i)(nick)GGCGCACGGAAUU (guide)

The results of this example are indicated in FIGS. 10A and 10B. As can be seen from this example, modifying the internal ends with one or more deoxyabasic moieties or modifications can impart further improvement of activity to the segmented miRNA mimetics of the invention.

Example 6

Segmented miRNA Mimetics Comprising Abasic Substitutions

Segmented miRNA mimetics can be designed to include a discontinuity comprising one or more non-nucleotide substitutions of the invention that occupy deleted sequence portions. In the following example, abasic linkers were used to substitute deleted nucleotide positions in both the guide and passenger strands of a miRNA mimetic. Likewise, substitution with other non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table VII), comprising one or more abasic substitutions and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICER^(ex5), a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692 (2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target genes, CD164 and VAMP3, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96).

The positions of the abasic linker is indicated as “(ab)” in both the names and the sequences of the following table. Passenger strand sequences in Table VII are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE VII  SEQ ID NO(s). Name Sequence P G miR-124 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 ACCGUAAGUGGCGCACGGAAUU (guide) (124p)8(ab)₂11/ (passenger) GCAUUCAC(ab)(ab)CGUGCCUUAAAU 1148 1149 (124g)9(ab)₂12 ACCGUAAGUGG(ab)(ab)CACGGAAUU (guide) (124p)7(ab)₃11/ (passenger) GCAUUCA(ab)(ab)(ab)CGUGCCUUAAAU 1150 1151 (124g)9(ab)₃13 ACCGUAAGUG(ab)(ab)(ab)CACGGAAUU (guide) (124p)/6(ab)₄11/ (passenger) GCAUUC(ab)(ab)(ab)(ab)CGUGCCUUAAAU 1152 1153 (124g)9(ab)₄14 ACCGUAAGU(ab)(ab)(ab)(ab)CACGGAAUU (guide) (124p)/5(ab)₅11/ (passenger) GCAUU(ab)(ab)(ab)(ab)(ab)CGUGCCUUAAAU 1154 1155 (124g)9(ab)₅15 ACCGUAAG(ab)(ab)(ab)(ab)(ab)CACGGAAUU (guide) (124p)/7(ab)₂10/ (passenger) GCAUUCA(ab)(ab)GCGUGCCUUAAAU 1148 1156 (124g)10(ab)₂13 ACCGUAAGUG(ab)(ab)GCACGGAAUU (guide) (124p)/6(ab)₃10/ (passenger) GCAUUC(ab)(ab)(ab)GCGUGCCUUAAAU 1157 1158 (124g)10(ab)₃14 ACCGUAAGU(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/5(ab)₄10/ (passenger) GCAUU(ab)(ab)(ab)(ab)GCGUGCCUUAAAU 1159 1160 (124g)10(ab)₄15 ACCGUAAG(ab)(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/4(ab)₅10/ (passenger) GCAU(ab)(ab)(ab)(ab)(ab)GCGUGCCUUAAAU 1161 1162 (124g)10(ab)₅16 ACCGUAA(ab)(ab)(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1163 8(ab)₂11 ACCGUAAGUGGC(ab)(ab)ACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1164 8(ab)₃12 ACCGUAAGUGG(ab)(ab)(ab)ACGGAAUU (guide) (124p)/(124g) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1165 8(ab)₄13 ACCGUAAGUG(ab)(ab)(ab)(ab)ACGGAAUU (guide) (124p)9(ab)11/ (passenger) GCAUUCACC(ab)CGUGCCUUAAAU 1166 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)8(ab)₂11/ (passenger) GCAUUCAC(ab)(ab)CGUGCCUUAAAU 1148 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)7(ab)₃11/ (passenger) GCAUUCA(ab)(ab)(ab)CGUGCCUUAAAU 1150 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)6(ab)₄11/ (passenger) GCAUUC(ab)(ab)(ab)(ab)CGUGCCUUAAAU 1152 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)5(ab)₅11/  (passenger) GCAUU(ab)(ab)(ab)(ab)(ab)CGUGCCUUAAAU 1154 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)4(ab)₆11/ (passenger) GCAU(ab)(ab)(ab)(ab)(ab)(ab)CGUGCCUUAAAU 1167 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)8(ab)10/ (passenger) GCAUUCAC(ab)GCGUGCCUUAAAU 1168 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)7(ab)₂10/ (passenger) GCAUUCA(ab)(ab)GCGUGCCUUAAAU 1169 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)6(ab)₃10/ (passenger) GCAUUC(ab)(ab)(ab)GCGUGCCUUAAAU 1170 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)5(ab)₄10/ (passenger) GCAUU(ab)(ab)(ab)(ab)GCGUGCCUUAAAU 1171 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)4(ab)₅10/ (passenger) GCAU(ab)(ab)(ab)(ab)(ab)GCGUGCCUUAAAU 1172 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)3(ab)₆10/ (passenger) GCA(ab)(ab)(ab)(ab)(ab)(ab)GCGUGCCUUAAAU 1173 1091 (124g) ACCGUAAGUGGCGCACGGAAUU (guide) (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1156 (ab)₂13 ACCGUAAGUG(ab)(ab)GCACGGAAUU (guide) (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1158 (ab)₃14 ACCGUAAGU(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1160 (ab)₄15 ACCGUAAG(ab)(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/(124g)10 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1162 (ab)₅16 ACCGUAA(ab)(ab)(ab)(ab)(ab)GCACGGAAUU (guide) (124p)/(124g)11 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1174 (ab)₂14 ACCGUAAGU(ab)(ab)CGCACGGAAUU (guide) (124p)/(124g)11 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1175 (ab)₃15 ACCGUAAG(ab)(ab)(ab)CGCACGGAAUU (guide) (124p)/(124g)11 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1176 (ab)₄16 ACCGUAA(ab)(ab)(ab)(ab)CGCACGGAAUU (guide) (124p)/(124g)11 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1177 (ab)₅17 ACCGUA(ab)(ab)(ab)(ab)(ab)CGCACGGAAUU (guide)

The results of this example are indicated in FIGS. 11A and 11B. As can be seen from this example, abasic substitutions can impart further advantageous properties to the segmented miRNA mimetics of the invention.

Example 7

Segmented miRNA Mimetics with Multiple Nucleotide Position Deletions and Substitutions

Segmented miRNA mimetics can be designed to include a discontinuity comprising one or more non-nucleotide substitutions of the invention that occupy deleted sequence portions of 1 or more nucleotide positions. In the following example, alkyl linkers were used to substitute deleted nucleotide positions in both the guide and passenger strands of a miRNA mimetic Likewise, substitution with other non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.

Oligonucleotides comprising C3 and C6 linkers were synthesized using protocols well known in the art (solid phase synthesis) using commercially available phosphoramidites, then purified by reversed phase solid phase extraction (SPE). The C3 (C₃₃H₄₃N₂O₅P) and C6 (C₃₆H₄₉N₂O₅P) phosphoramidites were purchased from ChemGenes.

Briefly, the single strand oligonucleotides were synthesized using phosphoramidite chemistry on an automated solid-phase synthesizer, using procedures as are generally known in the art (see for example U.S. application Ser. No. 12/064,014). A synthesis column was packed with solid support derivatized with the first nucleoside residue (natural or chemically modified). Synthesis was initiated by detritylation of the acid labile 5′-O-dimethoxytrityl group to release the 5′-hydroxyl. A suitably protected phosphoramidite and a suitable activator in acetonitrile were delivered simultaneously to the synthesis column resulting in coupling of the amidite to the 5′-hydroxyl. The column was then washed with a solvent, such as acetonitrile. An oxidizing solution, such as an iodine solution was pumped through the column to oxidize the phosphite triester linkage P(III) to its phosphotriester P(V) analog. Unreacted 5′-hydroxyl groups were capped using reagents such as acetic anhydride in the presence of 2,6-lutidine and N-methylimidazole. The elongation cycle was resumed with the detritylation step for the next phosphoramidite incorporation. This process was repeated until the desired sequence was synthesized. The synthesis concluded with the final 5′-terminus protecting group (trityl or 5′-O-dimethoxytrityl).

Upon completion of the synthesis, the solid-support and associated oligonucleotide were dried under argon pressure or vacuum. Aqueous base was added and the mixture was heated to effect cleavage of the succinyl linkage, removal of the cyanoethyl phosphate protecting group, and deprotection of the exocyclic amine protection.

The following process was performed on single strands that do not contain ribonucleotides. After treating the solid support with the aqueous base, the mixture was filtered to separate the solid support from the deprotected crude synthesis material. The solid support was then rinsed with DMSO, which is combined with the filtrate. The resultant basic solution allows for retention of the 5′-O-dimethoxytrityl group to remain on the 5′ terminal position (trityl-on).

For single strands that contain ribonucleotides, the following process was performed. After treating the solid support with the aqueous base, the mixture was filtered to separate the solid support from the deprotected crude synthesis material. The solid support was then rinsed with dimethylsulfoxide (DMSO), which was combined with the filtrate. Fluoride reagent, such as triethylamine trihydrofluoride, was added to the mixture, and the solution was heated. The reaction was quenched with suitable buffer to provide a solution of crude single strand with the 5′-O-dimethoxytrityl group on the final 5′ terminal position.

The trityl-on solution of each crude single strand was purified using chromatographic purification, such as SPE RPC purification. The hydrophobic nature of the trityl group permits stronger retention of the desired full-length oligo than the non-tritylated truncated failure sequences. The failure sequences were selectively washed from the resin with a suitable solvent, such as low percent acetonitrile. Retained oligonucleotides were then detritylated on-column with trifluoroacetic acid to remove the acid-labile trityl group. Residual acid was washed from the column, a salt exchange was performed, and a final desalting of the material commenced. The full-length oligo was recovered in a purified form with an aqueous-organic solvent. The final product was then analyzed for purity (HPLC), identity (Maldi-TOF MS), and yield (UV A260). The oligos were dried via lyophilization or vacuum condensation.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table VIII), from which bases have been deleted, and a non-targeting control “Universal Control (UC3)” duplex were transfected into HCT-116 cells (wild-type) and cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 6000 cells/well 24 hours prior to transfection. Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final concentration of our miRNAs at 10 nM. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CT™ Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using gene-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization. Knockdown of miR-124 targets was calculated as the two-fold change in target cDNA measured in experimentally-treated cells relative to the target cDNA measured in non-targeting control-treated cells.

The positions of deleted bases are indicated as “(

)” in both the names and the sequences of the following table. C3 and C6 linkers are identified as “(c3)” and “(c6)”, respectively. Passenger strand sequences in Table VIII are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE VIII SEQ ID NO(s). Name Sequence P G miR-124 (iP/G) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP9-10del/G11- (passenger) GCAUUCAC(

)(

)CGUGCCUUAAAU 1097 1093 12del ACCGUAAGUG(

)(

)GCACGGAAUU (guide) iP8-10del/G11- (passenger) GCAUUCA(

)(

)(

)CGUGCCUUAAAU 1097 1093 13del ACCGUAAGU(

)(

)(

)GCACGGAAUU (guide) iP7-10del/G11- (passenger) GCAUUC(

)(

)(

)(

)CGUGCCUUAAAU 1097 1093 14del ACCGUAAG(

)(

)(

)(

)GCACGGAAUU (guide) iP7-11del/G10- (passenger) GCAUUC(

)(

)(

)(

)(

)GUGCCUUAAAU 1178 n/a 14del ACCGUAAG(

)(

)(

)(

)(

)CACGGAAUU (guide) iP7-12del/G9- (passenger) GCAUUC(

)(

)(

)(

)(

)(

)UGCCUUAAAU 1109 n/a 14del ACCGUAAG(

)(

)(

)(

)(

)(

)ACGGAAUU (guide) iP9- (passenger) GCAUUCAC(

)(

)(c3)CGUGCCUUAAAU 1179 1180 10_1x_c3/G11- ACCGUAAGUG(

)(

)(c3)GCACGGAAUU (guide) 12_1x_c3 iP8- (passenger) GCAUUCA(

)(

)(

)(c3)CGUGCCUUAAAU 1181 1182 10_1x_c3/G11- ACCGUAAGU(

)(

)(

)(c3)GCACGGAAUU (guide) 13_1x_c3 iP7- (passenger) GCAUUC(

)(

)(

)(

)(c3)CGUGCCUUAAAU 1183 1184 10_1x_c3/G11- ACCGUAAG(

)(

)(

)(

)(c3)GCACGGAAUU (guide) 14_1x_c3 iP7- (passenger) GCAUUC(

)(

)(

)(

)(

)(c3)GUGCCUUAAAU 1185 1186 11_1x_c3/G10- ACCGUAAG(

)(

)(

)(

)(

)(c3)CACGGAAUU (guide) 14_1x_c3 iP7- (passenger) GCAUUC(

)(

)(

)(

)(

)(

)(c3)UGCCUUAAAU 1187 1188 12_1x_c3/G9- ACCGUAAG(

)(

)(

)(

)(

)(

)(c3)ACGGAAUU (guide) 14_1x_c3 iP9- (passenger) GCAUUCAC(

)(

)(c3)CGUGCCUUAAAU 1179 1189 10_1x_c3/G11- ACCGUAAGUG(

)(

)(c6)GCACGGAAUU (guide) 12_1x_c6 iP8- (passenger) GCAUUCA(

)(

)(

)(c3)CGUGCCUUAAAU 1181 1190 10_1x_c3/G11- ACCGUAAGU(

)(

)(

)(c6)GCACGGAAUU (guide) 13_1x_c6 iP7- (passenger) GCAUUC(

)(

)(

)(

)(c3)CGUGCCUUAAAU 1183 1191 10_1x_c3/G11- ACCGUAAG(

)(

)(

)(

)(c6)GCACGGAAUU (guide) 14_1x_c6 iP7- (passenger) GCAUUC(

)(

)(

)(

)(

)(c3)GUGCCUUAAAU 1185 1192 11_1x_c3/G10- ACCGUAAG(

)(

)(

)(

)(

)(c6)CACGGAAUU (guide) 14_1x_c6 iP7- (passenger) GCAUUC(

)(

)(

)(

)(

)(

)(c3)UGCCUUAAAU 1187 1193 12_1x_c3/G9- ACCGUAAG(

)(

)(

)(

)(

)(

)(c6)ACGGAAUU (guide) 14_1x_c6

The results of this example are shown in FIGS. 17A and 17B. A number of the segmented miRNA mimetic constructs of this example achieved a significant knockdown effect as compared to the non-segmented duplex miR-124.

Example 8

Segmented miRNA Mimetics Comprising Small Substitutions

Segmented miRNA mimetics can be designed to include a discontinuity comprising non-nucleotide substitutions of the invention that occupy deleted nucleotide positions. In the following example, C3 alkyl linkers were used to substitute deleted nucleotide positions in both the guide and passenger strand of a miRNA mimetic Likewise, substitution with other non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table IX), comprising C3 substitutions, and a non-targeting control “Universal Control (UC3)” duplex were transfected into HCT-116 cells (wild-type) and cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 6000 cells/well 24 hours prior to transfection. Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final concentration of our miRNAs at 10 nM. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CT™ Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using gene-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization. Knockdown of miR-124 targets was calculated as the two-fold change in target cDNA measured in experimentally-treated cells relative to the target cDNA measured in non-targeting control-treated cells.

The positions of c3 substitutions are shown in both the names and the sequences of the following table. C3 linkers are identified as “(c3)”. Passenger strand sequences in Table IX are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE IX  SEQ ID NO(s). Name Sequence P G miR-124 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 (iP/G) ACCGUAAGUGGCGCACGGAAUU (guide) iP22-22c3/G (passenger) GCAUUCACCGCGUGCCUUAAA(c3) 1194 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP21-21c3/G (passenger) GCAUUCACCGCGUGCCUUAA(c3)U 1195 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP20- (passenger) GCAUUCACCGCGUGCCUUA(c3)AU 1196 1216 20c3/G1-1c3 ACCGUAAGUGGCGCACGGAAU(c3) (guide) iP19- (passenger) GCAUUCACCGCGUGCCUU(c3)AAU 1197 1217 19c3/G2-2c3 ACCGUAAGUGGCGCACGGAA(c3)U (guide) iP18- (passenger) GCAUUCACCGCGUGCCU(c3)AAAU  1198 1218 18c3/G3-3c3 ACCGUAAGUGGCGCACGGA(c3)UU (guide) iP17- (passenger) GCAUUCACCGCGUGCC(c3)UAAAU 1199 1219 17c3/G4-4c3 ACCGUAAGUGGCGCACGG(c3)AUU (guide) iP16- (passenger) GCAUUCACCGCGUGC(c3)UUAAAU 1200 1220 16c3/G5-5c3 ACCGUAAGUGGCGCACG(c3)AAUU (guide) iP15- (passenger) GCAUUCACCGCGUG(c3)CUUAAAU 1201 1221 15c3/G6-6c3 ACCGUAAGUGGCGCAC(c3)GAAUU (guide) iP14- (passenger) GCAUUCACCGCGU(c3)CCUUAAAU 1202 1222 14c3/G7-7c3 ACCGUAAGUGGCGCA(c3)GGAAUU (guide) iP13- (passenger) GCAUUCACCGCG(c3)GCCUUAAAU 1203 1223 13c3/G8-8c3 ACCGUAAGUGGCGC(c3)CGGAAUU (guide) iP12- (passenger) GCAUUCACCGC(c3)UGCCUUAAAU 1204 1224 12c3/G9-9c3 ACCGUAAGUGGCG(c3)ACGGAAUU (guide) iP11-11c3/ (passenger) GCAUUCACCG(c3)GUGCCUUAAAU 1205 1225 G10-10c3 ACCGUAAGUGGC(c3)CACGGAAUU (guide) iP10-10c3/ (passenger) GCAUUCACC(c3)CGUGCCUUAAAU 1206 1226 G11-11c3 ACCGUAAGUGG(c3)GCACGGAAUU (guide) iP9-9c3/G12- (passenger) GCAUUCAC(c3)GCGUGCCUUAAAU 1207 1227 12c3 ACCGUAAGUG(c3)CGCACGGAAUU (guide) iP8-8c3/G13- (passenger) GCAUUCA(c3)CGCGUGCCUUAAAU 1208 1228 13c3 ACCGUAAGU(c3)GCGCACGGAAUU (guide) iP7-7c3/G14- (passenger) GCAUUC(c3)CCGCGUGCCUUAAAU 1209 1229 14c3 ACCGUAAG(c3)GGCGCACGGAAUU (guide) iP6-6c3/G15- (passenger) GCAUU(c3)ACCGCGUGCCUUAAAU 1210 1230 15c3 ACCGUAA(c3)UGGCGCACGGAAUU (guide) iP5-5c3/G16- (passenger) GCAU(c3)CACCGCGUGCCUUAAAU 1211 1231 16c3 ACCGUA(c3)GUGGCGCACGGAAUU (guide) iP4-4c3/G17- (passenger) GCA(c3)UCACCGCGUGCCUUAAAU 1212 1232 17c3 ACCGU(c3)AGUGGCGCACGGAAUU (guide) iP3-3c3/G18- (passenger) GC(c3)UUCACCGCGUGCCUUAAAU 1213 1233 18c3 ACCG(c3)AAGUGGCGCACGGAAUU (guide) iP2-2c3/G19- (passenger) G(c3)AUUCACCGCGUGCCUUAAAU 1214 1234 19c3 ACC(c3)UAAGUGGCGCACGGAAUU (guide) iP1-1c3/G20- (passenger) (c3)CAUUCACCGCGUGCCUUAAAU 1215 1235 20c3 AC(c3)GUAAGUGGCGCACGGAAUU (guide) iP/G21-21c3 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1236 A(c3)CGUAAGUGGCGCACGGAAUU (guide)

The results of this example are shown in FIGS. 18A and 18B. A number of the segmented miRNA mimetics of this example showed improved knockdown in comparison to the non-segmented duplex miR-124.

Example 9

Segmented miRNA Mimetics Comprising Larger Substitutions

Segmented miRNA mimetics can be designed to include a discontinuity comprising non-nucleotide substitutions of the invention that occupy deleted nucleotide positions. In the following example, C6 alkyl linkers were used to substitute deleted nucleotide positions in both the guide and passenger strand of a miRNA mimetic Likewise, substitution with other larger non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table X), comprising c6 substitutions, and a non-targeting control “Universal Control (UC3)” duplex were transfected into HCT-116 cells (wild-type) and cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 6000 cells/well 24 hours prior to transfection. Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final concentration of our miRNAs at 10 nM. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CT™ Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using gene-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization. Knockdown of miR-124 targets was calculated as the two-fold change in target cDNA measured in experimentally-treated cells relative to the target cDNA measured in non-targeting control-treated cells.

The positions of c6 substitutions are shown in both the names and the sequences of the following table. C6 linkers are identified as “(c6)”. Passenger strand sequences in Table X are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE X  SEQ ID NO(s). Name Sequence P G miR-124 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 (iP/G) ACCGUAAGUGGCGCACGGAAUU (guide) iP22-22c6/G (passenger) GCAUUCACCGCGUGCCUUAAA(c6) 1237 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP21-21c6/G (passenger) GCAUUCACCGCGUGCCUUAA(c6)U 1238 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP20- (passenger) GCAUUCACCGCGUGCCUUA(c6)AU 1239 1259 20c6/G1-1c6 ACCGUAAGUGGCGCACGGAAU(c6) (guide) iP19- (passenger) GCAUUCACCGCGUGCCUU(c6)AAU 1240 1260 19c6/G2-2c6 ACCGUAAGUGGCGCACGGAA(c6)U (guide) iP18- (passenger) GCAUUCACCGCGUGCCU(c6)AAAU 1241 1261 18c6/G3-3c6 ACCGUAAGUGGCGCACGGA(c6)UU (guide) iP17- (passenger) GCAUUCACCGCGUGCC(c6)UAAAU 1242 1262 17c6/G4-4c6 ACCGUAAGUGGCGCACGG(c6)AUU (guide) iP16- (passenger) GCAUUCACCGCGUGC(c6)UUAAAU 1243 1263 16c6/G5-5c6 ACCGUAAGUGGCGCACG(c6)AAUU (guide) iP15- (passenger) GCAUUCACCGCGUG(c6)CUUAAAU 1244 1264 15c6/G6-6c6 ACCGUAAGUGGCGCAC(c6)GAAUU (guide) iP14- (passenger) GCAUUCACCGCGU(c6)CCUUAAAU 1245 1265 14c6/G7-7c6 ACCGUAAGUGGCGCA(c6)GGAAUU (guide) iP13- (passenger) GCAUUCACCGCG(c6)GCCUUAAAU 1246 1266 13c6/G8-8c6 ACCGUAAGUGGCGC(c6)CGGAAUU (guide) iP12- (passenger) GCAUUCACCGC(c6)UGCCUUAAAU 1247 1267 12c6/G9-9c6 ACCGUAAGUGGCG(c6)ACGGAAUU (guide) iP11- (passenger)GCAUUCACCG(c6)GUGCCUUAAAU 1248 1268 11c6/G10-10c6 ACCGUAAGUGGC(c6)CACGGAAUU (guide) iP10- (passenger) GCAUUCACC(c6)CGUGCCUUAAAU 1249 1269 10c6/G11-11c6 ACCGUAAGUGG(c6)GCACGGAAUU (guide) iP9-9c6/G12- (passenger) GCAUUCAC(c6)GCGUGCCUUAAAU 1250 1270 12c6 ACCGUAAGUG(c6)CGCACGGAAUU (guide) iP8-8c6/G13- (passenger) GCAUUCA(c6)CGCGUGCCUUAAAU 1251 1271 13c6 ACCGUAAGU(c6)GCGCACGGAAUU (guide) iP7-7c6/G14- (passenger) GCAUUC(c6)CCGCGUGCCUUAAAU 1252 1272 14c6 ACCGUAAG(c6)GGCGCACGGAAUU (guide) iP6-6c6/G15- (passenger) GCAUU(c6)ACCGCGUGCCUUAAAU 1253 1273 15c6 ACCGUAA(c6)UGGCGCACGGAAUU (guide) iP5-5c6/G16- (passenger) GCAU(c6)CACCGCGUGCCUUAAAU 1254 1274 16c6 ACCGUA(c6)GUGGCGCACGGAAUU (guide) iP4-4c6/G17- (passenger) GCA(c6)UCACCGCGUGCCUUAAAU 1255 1275 17c6 ACCGU(c6)AGUGGCGCACGGAAUU (guide) iP3-3c6/G18- (passenger) GC(c6)UUCACCGCGUGCCUUAAAU 1256 1276 18c6 ACCG(c6)AAGUGGCGCACGGAAUU (guide) iP2-2c6/G19- (passenger) G(c6)AUUCACCGCGUGCCUUAAAU 1257 1277 19c6 ACC(c6)UAAGUGGCGCACGGAAUU (guide) iP1-1c6/G20- (passenger) (c6)CAUUCACCGCGUGCCUUAAAU 1258 1278 20c6 AC(c6)GUAAGUGGCGCACGGAAUU (guide) iP/G21-21c6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1279 A(c6)CGUAAGUGGCGCACGGAAUU (guide)

The results of this example are indicated in FIGS. 19A and 19B. A number of the segmented miRNA mimetics of this example showed increased knockdown in comparison to the non-segmented duplex miR-124.

Example 10

Segmented miRNA Mimetics Comprising Non-Nucleotide Insertions

Segmented miRNA mimetics can be designed to include a discontinuity comprising non-nucleotide insertions of the invention. In the following example, both small (C3) and larger (C6) alkyl moieties were used to connect segmented positions in both the guide and passenger strand of a miRNA mimetic. Likewise, insertions with other non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.

Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table XI), comprising c3 and c6 insertions, and a non-targeting control “Universal Control (UC3)” duplex were transfected into HCT-116 cells (wild-type) and cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 6000 cells/well 24 hours prior to transfection. Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final concentration of our miRNAs at 10 nM. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CT™ Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using gene-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization. Knockdown of miR-124 targets was calculated as the two-fold change in target cDNA measured in experimentally-treated cells relative to the target cDNA measured in non-targeting control-treated cells.

The positions of c3 and c6 insertions are shown in both the names and the sequences of the following table. C3 and C6 linkers are identified as “(c3)” and “(c6)”, respectively. Passenger strand sequences in Table XI are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.

TABLE XI  SEQ ID NO(s). Name Sequence P G miR-124 (iP/G) (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP/G1insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1280 ACCGUAAGUGGCGCACGGAAU(c6)U (guide) iP/G2insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1281 ACCGUAAGUGGCGCACGGAA(c6)UU (guide) iP/G3insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1282 ACCGUAAGUGGCGCACGGA(c6)AUU (guide) iP/G4insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1283 ACCGUAAGUGGCGCACGG(c6)AAUU (guide) iP/G5insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1284 ACCGUAAGUGGCGCACG(c6)GAAUU (guide) iP/G6insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1285 ACCGUAAGUGGCGCAC(c6)GGAAUU (guide) iP/G7insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1286 ACCGUAAGUGGCGCA(c6)CGGAAUU (guide) iP/G8insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1287 ACCGUAAGUGGCGC(c6)ACGGAAUU (guide) iP/G9insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1288 ACCGUAAGUGGCG(c6)CACGGAAUU (guide) iP/G10insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1289 ACCGUAAGUGGC(c6)GCACGGAAUU (guide) iP/G11insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1290 ACCGUAAGUGG(c6)CGCACGGAAUU (guide) iP/G12insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1291 ACCGUAAGUG(c6)GCGCACGGAAUU (guide) iP/G13insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1292 ACCGUAAGU(c6)GGCGCACGGAAUU (guide) iP/G14insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1293 ACCGUAAG(c6)UGGCGCACGGAAUU (guide) iP/G15insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1294 ACCGUAA(c6)GUGGCGCACGGAAUU (guide) iP/G16insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1295 ACCGUA(c6)AGUGGCGCACGGAAUU (guide) iP/G17insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1296 ACCGU(c6)AAGUGGCGCACGGAAUU (guide) iP/G18insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1297 ACCG(c6)UAAGUGGCGCACGGAAUU (guide) iP/G19insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1298 ACC(c6)GUAAGUGGCGCACGGAAUU (guide) iP/G20insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1299 AC(c6)CGUAAGUGGCGCACGGAAUU (guide) iP/G21insertc6 (passenger) GCAUUCACCGCGUGCCUUAAAU 1092 1300 A(c6)CCGUAAGUGGCGCACGGAAUU (guide) iP10insertc6/G (passenger) GCAUUCACCG(c6)CGUGCCUUAAAU 1301 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP9insertc6/G (passenger) GCAUUCACC(c6)GCGUGCCUUAAAU 1302 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP8insertc6/G (passenger) GCAUUCAC(c6)CGCGUGCCUUAAAU 1303 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP7insertc6/G (passenger) GCAUUCA(c6)CCGCGUGCCUUAAAU 1304 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP6insertc6/G (passenger) GCAUUC(c6)ACCGCGUGCCUUAAAU 1305 1091 ACCGUAAGUGGCGCACGGAAUU (guide) iP10insertc6/ (passenger) GCAUUCACCG(c6)CGUGCCUUAAAU 1301 1289 G10insertc6 ACCGUAAGUGGC(c6)GCACGGAAUU (guide) iP9insertc6/ (passenger) GCAUUCAC(c6)CGCGUGCCUUAAAU 1302 1290 G11insertc6 ACCGUAAGUG(c6)GCGCACGGAAUU (guide) iP8insertc6/ (passenger) GCAUUCA(c6)CCGCGUGCCUUAAAU 1303 1291 G12insertc6 ACCGUAAGU(c6)GGCGCACGGAAUU (guide) iP7insertc6/ (passenger) GCAUUC(c6)ACCGCGUGCCUUAAAU 1304 1292 G13insertc6 ACCGUAAG(c6)UGGCGCACGGAAUU (guide) iP6insertc6/ (passenger) GCAUU(c6)CACCGCGUGCCUUAAAU 1305 1293 G14insertc6 ACCGUAA(c6)GUGGCGCACGGAAUU (guide) iP10insertc3/ (passenger) GCAUUCACCG(c3)CGUGCCUUAAAU 1306 1307 G10insertc3 ACCGUAAGUGGC(c3)GCACGGAAUU (guide)

The results of this example are indicated in FIGS. 20A and 20B. A number of the segmented miRNA mimetics of this example showed increased knockdown in comparison to the non-segmented duplex miR-124.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. 

1. A double-stranded miRNA mimetic molecule represented by Formula III:

wherein (a) the molecule comprises a passenger strand and a guide strand where each line and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2” and “X3” represent the number of nucleotides in each stretch, “G/N” represents a discontinuity in the guide strand, “Y1” represents a number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present, and each of “Z1” and “Z2” represents the number of overhanging nucleotides (b) X1 is an integer from 16 to 26, X2 is an integer from 2 to 20, X3 is an integer from 6 to 24, Y1 is an integer from 0 to 6, provided that the sum of X2, X3 and Y1 is an integer from 16 to 26; Zi and Z2 are independently integers from 0 to 4; (c) N_(X3) comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence having any of SEQ ID NOs: 1-1090; (d) N_(X2) and N_(X3) together comprise sequence having at least 50% homology to the miRNA sequence; and (e) N_(X1) comprises sequence having at least 50% complementary to the miRNA sequence.
 2. The miRNA mimetic molecule of claim 1, wherein G/N comprises a nick in the guide strand.
 3. The miRNA mimetic molecule of claim 1, wherein G/N comprises one or more gap(s) in the guide strand.
 4. The miRNA mimetic molecule of claim 1, wherein G/N comprises one or more non-nucleotide substitutions in the guide strand.
 5. The miRNA mimetic molecule of claim 1, wherein G/N comprises one or more non-nucleotide insertions in the guide strand.
 6. The miRNA mimetic molecule of claim 1, wherein NX3 comprises a sequence having at least 7 contiguous nucleotides of the seed sequence of the miRNA.
 7. The miRNA mimetic molecule of claim 1, wherein NX3 comprises a sequence having at least 8 contiguous nucleotides of the seed sequence of the miRNA.
 8. The miRNA mimetic molecule of claim 1, wherein NX2 and NX3 together comprise a sequence having at least 60, 70, 80, or 90% homology to the miRNA sequence.
 9. The miRNA mimetic molecule of claim 1, wherein NX1 comprises a sequence having at least 60, 70, 80, or 90% complementarity to the miRNA sequence. 10-19. (canceled)
 20. A composition comprising the miRNA mimetic molecule of claim 1 and a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, or preservative. 